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A Kilejian

Publications and source records attributed to A Kilejian.

At least 19 recordsLinked to original sources
PubMed1991

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.

Actins↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1988

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.

Animals↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1986

Histidine-rich domain of the knob protein of the human malaria parasite Plasmodium falciparum.

Membranes of erythrocytes infected with the human malaria parasite Plasmodium falciparum develop protrusions called knobs. These structures are essential for the survival of the parasite in the host, and their induction requires the synthesis of the knob protein by the parasite. We describe the isolation of a cDNA clone encoding the amino-terminal half of the knob protein. A cDNA library was constructed from RNA prepared from ring stages of a P. falciparum isolate that has retained its ability to induce knobs (knob+ phenotype). A synthetic oligonucleotide probe encoding polyhistidine was used to isolate the cDNA clone, which encodes the amino-terminal half of a polypeptide with all the known attributes of the knob protein. The gene is not transcribed in variants that do not synthesize the knob protein and thereby cannot induce knobs (knob- phenotype). The apparent lack of transcription in knob- variants is due to different mechanisms: although the gene is present in one knob- isolate, it has been deleted in a cloned knob- variant. The primary structure of the polypeptide deduced from a partial sequence of the cDNA is distinctly different from other malarial histidine-rich polypeptides. The amino-terminal sequence shows the characteristic features of a signal peptide. This is followed by a histidine-rich domain and a subsequent region which contains one histidine. Peptide map analysis of the knob protein is consistent with the structural features deduced from the sequence analysis of the cDNA.

Amino Acid Sequence↗
PubMed1985

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.

Animals↗
PubMed1984

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.

Animals↗
PubMed1983

Immunological cross-reactivity of the histidine-rich protein of Plasmodium lophurae and the knob protein of Plasmodium falciparum.

Rabbits were immunized with the histidine-rich protein (HRP) of the avian malaria parasite, Plasmodium lophurae. The anti-HRP immunoprecipitated the knob protein (KP) from extracts of the human parasite, Plasmodium falciparum. The anti-HRP did not react with any antigens of a laboratory-derived strain of P. falciparum (K-) that does not form knobs. Antisera raised against a membrane-enriched fraction of P. falciparum-infected erythrocytes as well as sera from humans exposed to P. falciparum infections immunoprecipitated HRP from P. lophurae extracts.

Animals↗
PubMed1982

The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae.

Preliminary studies had indicated the inadequacy of the wheat germ and rabbit reticulocyte cell-free translation systems for the in vitro translation of mRNA isolated from Plasmodium lophurae. To identify the factors which are important for the efficient translation of parasite proteins, an homologous system was established using polysomes, the pH 5 fraction, and tRNA prepared from p. lophurae. For comparison, the same components were isolated from the host duck reticulocytes and tested. The effect of each of these factors was evaluated by analysis of the translation products and by comparison with products synthetized in vivo. The results indicated that P. lophurae tRNA had a marked stimulatory effect on the synthesis of parasite proteins while it inhibited the synthesis of host proteins. Duck reticulocyte tRNA could not be used as a substitute for the parasite tRNA. Based on these findings, a commercially available rabbit reticulocyte system was supplemented with P. lophurae tRNA, which markedly increased the efficiency of translation of P. lophurae proteins by this system.

Animals↗
PubMed1982

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum.

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.

Adenosine Triphosphate↗
PubMed1982

Comparative studies of antigens of erythrocytic and exoerythrocytic merozoites of Plasmodium lophurae: characterization of a surface glycoprotein from exoerythrocytic merozoites.

Rabbits were immunized with merozoite-enriched preparations of erythrocytic and exoerythrocytic Plasmodium lophurae. The antisera were used to compare antigens of the two types of merozoites. The indirect immunofluorescent antibody test showed the presence of common antigens. The growth of exoerythrocytic parasites was inhibited by the homologous antiserum and to a lesser extent by the antiserum prepared against erythrocytic forms. Cultures of exoerythrocytic parasites as well as their normal host cells were labeled metabolically with 35S-methionine, tritiated proline and glucosamine. Nonidet P-40 extracts of labeled merozoite-enriched preparations, infected cells, and normal cells were immunoprecipitated with the two types of antisera and the immunoprecipitates were analyzed on polyacrylamide gels. The results showed that erythrocytic and exoerythrocytic merozoites have several common proteins. A major difference was a glycoprotein with an approximate molecular weight of 110,000 daltons. This glycoprotein was associated with the surface of exoerythrocytic merozoites and was not recognized by antibodies prepared against erythrocytic forms.

Animals↗
PubMed1980

Homology between a histidine-rich protein from Plasmodium lophurae and a protein associated with the knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum.

The incorporation of several radioactive amino acids into the knob protein of Plasmodium falciparum was compared. Histidine showed better incorporation than proline. A protein hydrolysate, which had all major amino acids except histidine and methionine, showed relatively poor incorporation as compared with proline, and no labeling could be detected with methionine or leucine. These results strongly suggest that the amino acid composition of the knob protein has the same peculiarities as that of a histidine-rich protein characterized from P. lophurae. Immunoelectron microscopy suggested possible immunological cross-reactivity between these two proteins.

Animals↗