Plasmodium lophurae: Membrane Proteins of Erythrocyte-free Plasmodia and Malaria-Infected Red Cells*

SYNOPSIS. Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitrogen decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm³, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm³ and 1.158 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230,000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Abnormal bovine erythrocyte membrane proteins and glycoproteins during and after infection with Anaplasma marginale

The erythrocyte membrane proteins from normal and Anaplasma-infected bovine blood have been compared. Two distinct new polypeptides were present in membranes from acutely infected cells. The glycoprotein pattern was also altered: in addition to the three main bands observed in normal cells, there were four new bands present which were glycosylated. The normally found membrane glycolypeptide (250000 D) was missing. The role of these protein alterations in relation to the infectious process is discussed.     

Comparison of the Surface Proteins and Glycoproteins On Erythrocytes of Calves Before and During Infection With Babesia Bovis

The surface proteins and glycoproteins on red cells from normal and Babesia bovis-infected calf blood have been compared. Several radiolabeling probes were used to label specifically external membrane molecules which were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography or fluorography. No differences were observed among the Coomassie Blue-stained membrane proteins of erythrocytes from individual uninfected calves. Comparison of red cells from these animals also indicated no qualitative differences in the surface proteins with accessible tyrosyl residues labeled by lactoperoxidase-catalyzed radioiodnation, although some quantitative variation in the uptake of radioactivity into particular proteins was observed. the major radioiodinated bands on normal bovine erythrocytes had Mr of 165, 130, 90, and 45 kiloDaltons. However, labeling of surface glycoproteins by the periodate/[³H]NaBH₄ and galactose oxidase (± neuraminidase)/[³H]NaBH₄ methods showed significant differences in the surface proteins of red cells from individual uninfected calves. of 14 animals tested, 5 had major labeled glycoproteins of unique Mr. No changes were observed in radioiodinated surface proteins of total red cell samples from infected calves with 0.5-6% parasitemia. Radioiodination of concentrated infected red cells from the same samples (concentrated by selective hypotonic lysis of uninfected erythrocytes in KC1) resulted in the labeling of 3 new surface proteins, with Mr of 118, 115, and 60 kiloDaltons. the same new ¹²⁵I-labeled bands were identified on infected cells from 3 avirulent strains of B. bovis used in vaccine production. Furthermore, in concentrated infected cells there was very poor radiolabeling of major bands strongly labeled on uninfected cells (Mr 165, 130, and 90 kiloDaltons), suggesting parasite-induced loss of these proteins. Although there were some differences in ³H-labeled surface glycoproteins of red cells from normal and. B. bovis -infected blood, they were restricted to minor labeled bands and were not seen consistently. the labeled surface glycoproteins of concentrated infected cells were very similar to those of the uninfected red blood cells from infected blood.     

Differences in erythrocyte membrane proteins and glycoproteins in sickle cell disease

Erythrocyte membrane proteins obtained from individuals with sickle cell anemia show an SDS polyacrylamide gel pattern that differs in five regions from the normal pattern. These membranes when compared with membranes from normal individuals also show a marked decrease in sialic acid content which correlates with a marked reduction of the periodic acid-Schiff staining of the three major glycoprotein components. The observed membrane protein and glycoprotein changes are a characteristic of all the red cells in sickle cell anemia and do not correlate with the proportion of irreversibly sickled cells.     

Total sialic acid content of glycophorins during senescence of human red blood cells.

Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.     

Red cell membrane in hemolytic disease Studies on variables affecting electrophoretic analysis

Significant alterations in the spectrin: band 3 and band 4.1a : band 4.1b ratios and an occasional decrease in the peak height of band 4.2 with respect to band 4.1 were found in electrophoretic patterns of red cell membranes from patients with hereditary xerocytosis. Electrophoretic comparison of whole cell, cytoplasm and membrane polypeptides implied that atypical partitioning at hemolysis could account for some, but not all, of the alterations seen in membrane patterns of xerocytes. A decrease in band 4.2 peak height as well as a variation in the profile of band 3 were produced in controls by specific manipulations of the electrophoresis protocol. Metabolic depletion of normal cells produced the type of alterations in bands 3 and 4.1 found in xerocyte membranes, whereas Heinz body production, addition of calcium to the hemolysis buffer and incubation of membranes in detergent under conditions designed to promote proteolysis did not. The presence of a higher peak height of band 4.1b with respect to that of band 4.1a in membranes of patients with various other red cell disorders correlated with an increase in the percentage of reticulocytes in peripheral circulation. The appearance of both band 3 and 4.1 abnormalities in the patterns of control cells which had been enriched in young cells by density gradient centrifugation suggested that these alterations in hemolytic disease are related to the predominance of young cells in the population.     

Red cell membrane in hemolytic disease. Studies on variables affecting electrophoretic analysis

Significant alterations in the spectrin: band 3 and band 4.1a: band 4.1b ratios and an occasional decrease in the peak height of band 4.2 with respect to band 4.1 were found in electrophoretic patterns of red cell membranes from patients with hereditary xerocytosis. Electrophoretic comparison of whole cell, cytoplasm and membrane polypeptides implied that atypical partitioning at hemolysis could account for some, but not all, of the alterations seen in membrane patterns of xerocytes. A decrease in band 4.2 peak height as well as a variation in the profile of band 3 were produced in controls by specific manipulations of the electrophoresis protocol. Metabolic depletion of normal cells produced the type of alterations in bands 3 and 4.1 found in xerocyte membranes, whereas Heinz body production, addition of calcium to the hemolysis buffer and incubation of membranes in detergent under conditions designed to promote proteolysis did not. The presence of a higher peak height of band 4.1b with respect to that of band 4.1a in membranes of patients with various other red cell disorders correlated with an increase in the percentage of reticulocytes in peripheral circulation. The appearance of both band 3 and 4.1 abnormalities in the patterns of control cells which had been enriched in young cells by density gradient centrifugation suggested that these alterations in hemolytic disease are related to the predominance of young cells in the population.     

Elution and uncoating of Coxsackievirus B3 by isolated HeLa cell plasma membranes.

Plasma membranes isolated from HeLa cells on discontinuous sucrose gradients were assayed for their capacity to elute and uncoat coxsackievirus B3 at 37 C. Because the viral receptors are limited to the surface of HeLa cells, the addition of radioactively labeled virus to the cells prior to cell homogenization provided a useful marker for locating the plasma membranes during the fractionation procedure. Four bands were formed on the discontinuous sucrose gradients with approximately 70% or more of the membrane-associated viral label being recovered in the most dense bands, designated as bands 3 and 4. Bands 3 and 4 also possessed the plasma membrane marker enzymes, Na+, K+ adenosine triphosphatase and 5'-nucleotidase and revealed typical structures characteristic of plasma membranes as revealed by electron microscopy. Pelleted and washed membranes from band 3 both eluted and uncoated B3 32P-labeled virus, whereas membranes from band 4 eluted virus but failed to uncoat it. The membranes from band 4 were shown to inhibit the viral uncoating activity when mixed with membranes of band 3. Characteristically, unfractionated homogenates of cell membranes eluted but did not uncoat virus. The finding of a naturally occurring inhibitor of virus uncoating provides for the first time a way to distinguish between the membrane activities of virus elution and virus uncoating. The inhibitor remains to be characterized.     

Proteins specified by herpes simplex virus. XIII. Glycosylation of viral polypeptides.

In the course of herpes simplex virus 1 (HSV-1) replication in human epidermoid carcinoma no. 2 cells, the synthesis and glycosylation of host cell proteins ceases and is replaced by the synthesis and glycosylation of virus-specified polypeptides. Analyses of the synthesis of viral glycoproteins show that the glycosylation of viral polypeptides occurs late in the virus growth cycle and that certain of the precursors to major vital glycoproteins are members of the gamma group of polypeptides, i.e., polypeptides synthesized at increasing rates until 12 to 15 h postinfection. Viral glycoproteins are formed by stepwise additions of heterosaccharide chains to completed precursor polypeptides. The precursor and the highly glycosylated product are separable by gel electrophoresis and are localized in different fractions of infected cells. Within 15 min of their synthesis, precursor polypeptides acquire heterosaccharide chains of about 2,000 molecular weight, which contain glucosamine but little or nor fucose or sialic acid. Both precursor and product of this first stage of glycosylation are absent or present in low concentrations in the surface membranes of the infected cell and in the virion. The partially glycosylated product is then conjugated further in a slow, discontinuous process to form the mature glycoprotein of the virion and plasma membrane. These mature products bear large heterosaccharide units with molecular weights greater than 4,000 to 5,000; these contain fucose and sialic acid as well as glucosamine. Heterosaccharide chains from infected and uninfected cells are distributed among discrete size classes and the smallest chains consist of multiple saccharide residues.     

Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes

Malaria parasites during intraerythrocytic development change the ultrastructure, biophysics, and the antigens of the host red blood cell membrane. Parasite-encoded proteins are associated with, inserted into, or secreted across the infected erythrocyte membrane. Since parasites of the genus Plasmodium are eukaryotic cells, it must be assumed that they possess essentially eukaryotic modes of vesicle-mediated transport and translocation of proteins and membranes. Numerous studies have demonstrated vesicular structures in the cytoplasm of malaria-infected red blood cells and an assortment of parasite proteins associated with the different vesicles, membranes, and membrane-defined compartments. Some parasite polypeptides remain trapped between the parasite and the parasitophorous vacuole membranes PVM, whereas others are associated with morphologically distinct membrane-limited vesicles and vacuoles. Some of these same parasite protein antigens also associate with the erythrocyte membrane or with parasite-induced ultrastructural modifications in the membrane of the parasitized red blood cells. This implies that intracellular transport occurs in malaria-infected erythrocytes, a capacity that uninfected red blood cells normally lose upon enucleation. The specific locations of parasite antigens within the infected cell also implys the existence of targeting signals in the translocated parasite polypeptides and perhaps transport-mediating proteins. The genes corresponding to some of these translocated proteins have been sequenced. Typical (and in some cases atypical) signal peptide sequences occur, as well as a number of sequences that may result in posttranslational modifications. How or if these features figure in to the translocation across, and targeting to a particular membrane compartment of the intraerythrocytic parasite remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.     

An Electron Microscope-Cytochemical Method for Differentiating Membranes of Host Red Cells and Malaria Parasites*

Limiting membranes of malaria parasites and host red cells stain differently when exposed to positively charged iron colloid. Negatively charged red cell membranes avidly bind colloid, whereas parasite membranes do not. This selectivity in colloidal iron uptake by the 2 types of membranes can be utilized as an aid in discerning the amounts of contaminating host cell membranes in “free” malaria parasite preparations and in related cell-free membrane extracts.     

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.     

Alterations in membrane proteins of mouse erythrocytes infected with different species and strains of malaria parasites.

1. The membrane fraction, prepared by hypotonic lysis, of mouse red cells infected with Plasmodium berghei, P. yoelii YM, P. yoelii 17 X, P. yoelii 33 X, P. vinckei or P. chabaudi shows significant alterations from normal in protein composition as observed by dodecylsulphate-polyacrylamide gel electrophoresis. 2. There is a reduction in intensity of various protein bands, notably bands I and II (spectrin), of membranes prepared from infected red cells. 3. New bands are observed as a result of infection, the intensity and location of which depend on the parasite species and strain. A new band of apparent molecular weight 150,000 appears with a strong intensity in P. yoelii YM infection, with a moderate intensity in P. berghei infection, and with a weak intensity in P. vinckei and P. chabaudi infection. In P. yoelii 17X and 33X infection, multiple weak bands are seen in the molecular weight range 120,000-210,000.     

Isolation and characterization of the erythrocyte surface membrane of the smooth dogfish, Mustelus canis.

1. The plasma membrane of the dogfish erythrocyte is characterized. 2. Surface membranes were isolated from dogfish red cells. 3. Cells suspended in a hypotonic zinc chloride buffer (0.5 mM ZnCl2, 5 mM Tris-HCl, pH 8.0) tended to enucleate spontaneously. 4. By gentle homogenization relatively high yields of red cell "ghosts" were formed; these were purified using discontinuous gradients of sucrose and of glycerol solutions. Analyses of protein, carbohydrates and lipids were carried out. 5. There did not appear to be marked overall differences in the composition of the surface membrane of the nucleated dogfish erythrocyte compared to those of other species.     

Plasmodium falciparum exported protein PFE60 influences Maurer’s clefts architecture and virulence complex composition

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer’s clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore – unlike all other described resident MC membrane proteins – PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.     

Various topochemical arrangements of sialic acids on human erythrocytes as detected by partition in aqueous two-polymer phase systems

Human and rabbit red blood cells were subjected to partition in an aqueous, buffered Ficoll-Dextran two-phase system. The effect of neuraminidase treatment on the cell partition behaviour was examined and compared with the amount of sialic acids released from the cell surface and with the change in the electrophoretic mobility of the cells. The data obtained in the study indicate that there are two main groups of sialic acids differing in their topochemical arrangement on the human erythrocyte surface, and their relative hydrophobicity was evaluated. The results obtained in the case of rabbit red cells seem to indicate that sialic acids present on the cell surface are not the only major ionogenic surface components as is the case for human red cells. The data obtained support the assumption that the membrane surface charge is the determinant of cell partition only as a factor affecting the relative hydrophobicity of the cell surface.     

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.