Isolation and characterization of the plasma membrane of human erythrocytes infected with the malarial parasite Plasmodium falciparum.

Abstract

Human erythrocytes infected with the malarial parasite Plasmodiumfalciparum were labeled metabolically with a mixture of 15 radioactive amino acids. When synchronously growing parasites were at the schizont stage of development infected cells were concentrated and purified by using a PercollHypaque gradient. The plasma membrane of the infected erythrocyte, isolated by binding cells to a solid support (Affi-Gel 731, Bio-Rad), was <1% contaminated with parasite membranes.Erythrocyte membrane proteins were analyzed by polyacrylamide gel electrophoresis and autoradiography. Despite the high sensitivity of the procedure, there was no evidence for the insertion of parasite proteins into the infected host cell membrane. One possible exception is a Mr 230,000 parasite protein present maximally as 9,000 copies per infected erythrocyte membrane. Moreover, no differences in the membrane proteins were observed between a highly knobby clone and a knobless clone of the same strain of P. falciparum. These findings appear to rule out the presence of parasite protein(s) playing a structural role in the formation of knobs on the erythrocyte surface and question whether the antigenic determinants on the P. falciparum-infected erythrocyte are of parasite origin or whether such antigens represent newly exposed or chemically modified erythrocyte determinants.The surface of the human erythrocyte infected with the late stages of asexual development of Plasmodiumfalciparum is morphologically altered by the presence of excrescences called knobs (1). The knobs bind ferritin-labeled antibodies derived from immune monkey sera (2) or sera raised against "purified" parasites (3). Such antigens are believed to represent parasite proteins inserted into the host cell membrane, in part because a Mr 80,000 protein of parasite origin was reported to be associated with the formation of knobs (4).To determine whether parasite proteins are, or are not, associated with the host cell membrane, the membrane of human erythrocytes containing radiolabeled P.falciparum was isolated and purified by using polyacrylamide beads coated with polyethylenimine. Such membranes were analyzed by gel electrophoresis and autoradiography or fluorography. Both a K' clone (high density of knobs on the infected erythrocyte surface) and a K0 clone (absence of knobs) of the same strain were used. MATERIALS AND METHODSIsolation of Schizont-Infected Erythrocytes. Two clones (K+ and K0) of P.falciparum, derived from the FCR-3 (Gambia) strain, were kindly provided by L. H. Miller (National Institutes of Health, Bethesda, MD). The parasites were grown according to Trager and Jensen (5) and synchronized at the ring stage according to Lambros and Vanderberg (6). When the parasitemia was 4% and the parasites were at the trophozoite stage, the cells were labeled metabolically for 12 hr by the addition of 3.7 X 105 becquerels per ml of medium with a mixture of 15 L amino acids. Because no protein synthesis takes place in mature erythrocytes, all of the radioactivity incorporate...

tRNA epitranscriptomics and biased codon are linked to proteome expression in Plasmodium falciparum

“…Given the nearly complete lack of information about RNA modifications in malaria parasites, we first identified the complete spectrum of modified ribonucleosides in tRNA from P. falciparum . As outlined in the workflow in Appendix Fig S1A, care was taken to selectively rupture RBC membranes with saponin to purify parasites (Gruenberg & Sherman, ) and then extract total RNA. Bioanalyzer analysis (Appendix Fig S1B) revealed tRNA and 5S, 5.8S, 18S, and 28S rRNAs from P. falciparum and the apicoplast organelle, with minor amounts of human 18S and 28S rRNA, which confirmed limited host cell RNA contamination.…”

Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein.

“…Cultures were synchronized at the ring stage by sorbitol lysis of mature forms (Lambros and Vanderberg, 1980). Mature forms of the parasite were concentrated by the method of Gruenberg and Sherman (1983).…”

Developmental modulation of protein synthetic patterns by the human malarial parasite Plasmodium falciparum

“…We found no developed for P. falciparurn (see Ref. 30). Therefore, evidence for removal of radiolabeled polypeptides from we attempted to localize any parasite-synthesized pro-the surface of intact red cells, regardless of the age of the teins on the red cell surface by modifying the surface parasites within or whether labeled with [14~]histidine Can.…”