The synthesis and fate of stage-specific proteins in Plasmodium falciparum cultures

Cultured ring, trophozoite and schizont stages of Plasmodium falciparum were metabolically labeled with [35S]methionine. After labeling, cultures were incubated for varying times in the presence of non-radioactive methionine. Triton-soluble proteins from different stages of growth were analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Most proteins were synthesized by every stage of growth and remained unchanged throughout the cycle through to the ring stage following merozoite invasion of erythrocytes. At least 15 proteins, most of high molecular weight, were synthesized solely or predominantly by schizonts. Eight proteins (approx. 177, 170, 158, 87, 83, 47, 41 and 24 kDa) appeared in schizonts but not merozoites. Eight proteins (approx. 240, 203, 106, 80, 35, 19, 15 and 14 kDa) appeared in merozoites, but not in rings following merozoite invasion. Some proteins appeared to be modified after synthesis.     

Two novel phospholipid-linked mouse thymocyte surface molecules released by phosphatidylinositol-specific phospholipase C

We searched for mouse thymocyte surface proteins attached to the cell membrane through a phosphatidylinositol (PI)-containing glycolipid similar to that identified in the T cell-activating Thy-1 glycoprotein. Our approach was to biochemically analyse the supernatants of ¹²⁵I surface-labeled thymocytes treated with 60 U/ml of Staphylococcus aureus PI-specific phospholipase C (PI-PLC). In addition to Thy-1, two molecules of M_r, 13,000 and 52,000 were found to be specifically solubilized by the enzymatic treatment. The 52,000 structure is a single basic polypeptide of M_r, 50,000 under non-reducing conditions. Two-dimensional gel electrophoresis analyses resolved the 13,000 mol. wt molecules in three relatively basic components including (i) a monomeric molecule(s), a fraction of which exhibited slower migration in reducing gels, and (ii) disulfide-linked multimeric structures comprising a major component of M_r, 30,000 and a minor one of M_r, 45,000. These 52,000 and 13,000 mol. wt molecules could be released from thymocytes and Escherichia coli lipopolysaccharide (LPS)-stimulated B cell blasts, but not from a variety of mature T cell populations. These data add new members to the list of PI-linked rodent lymphoid cell differentiation markers, which already includes three activation signal-transducing T cell molecules (i.e. Thy-1, Ly-6-linked T cell-activating proteins, and RT-6).     

Heterogeneity in the B1 (CD20) cell surface molecule expressed by human B-lymphocytes

The B1 molecule (CD20) is a phosphoprotein expressed only by B-lymphocytes. In this study. analysis of B1 immunoprecipitated from surface iodinated B-cell lines and B-lymphocytes has shown that there are several expressed forms of Bl. A predominant species of M_r 33,000 represents 75–80% of the iodinated cell surface B1 and a M_r 35,000 species represents 20–25%. Limited proteinase digestion of these two species generated similar peptide maps demonstrating that the different forms of B1 shared common peptides. Biosynthetic labeling with [³⁵S]methionine revealed that the M_r 35,000 B1 species may actually represent two bands of M_r 34,500 and 36,000. Endoglycosidase digestion studies and metabolic labeling in the presence of tunicamycin indicated that neither the M_r 33,000 or 34,500–36,000 forms of B1 were glycosylated. The M_r 33,000 and 34,500–36,000 forms of B1 were constitutively phosphorylated in B-cell lines. However, exposure of B-cells to PMA resulted in a significant increase in the phosphorylation of the M_r 34,500–36,000 form. Exposure to PMA also resulted in an increase in the amount of M_r 34,500–36,000 protein immunoprecipitated from ³⁵S labeled cells. These results suggest that there are multiple forms of the B1 molecule expressed by B-lymphocytes and that this heterogeneity may result from phosphorylation of the B1 protein.     

Equine infectious anemia virus, a putative lentivirus, contains polypeptides analogous to prototype-C oncornaviruses

The polypeptide composition of purified radioactive leucine or glucosamine-labeled equine infectious anemia virus (EIAV) was investigated using guanidine hydrochloride gel filtration (GHCI-GF) and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to Friend murine leukemia virus (FLV), a prototype-C oncornavirus. The apparent molecular weights and relative abundance of each EIAV polypeptide were calculated. The results of these studies indicate that EIAV contains four major nonglycosylated proteins (p26, p15, p11, and p9) and two glycoproteins (gp90 and gp45), which together account for greater than 95% of the total virion protein. Four minor polypeptides of unknown significance were also detected reproducibly. EIAV gp90 appears to be the more heavily glycosylated polypeptide, while the gp45 component aggregates in 6 M GHCI, evidently reflecting a hydrophobic character. No disulfide linkages were detected between the EIAV glycoproteins. These observations demonstrate for the first time that EIAV contains polypeptides analogous to prototype-C oncornaviruses, such as FLV. However, the demonstrated serological unrelatedness between EIAV and FLV was reflected in biochemical differences in protein apparent molecular weights and by the resistance of EIAV nonglycosylated proteins to dissociation in GHCI, a property shared by visna virus.     

Membrane glycoproteins of human polymorphonuclear leukocytes that act as receptors for mannose-specific Escherichia coli.

Type 1 fimbriated (mannose-specific) Escherichia coli cells bind to mannose residues on human polymorphonuclear leukocytes (PMN); this leads to phagocytosis of the bacteria. To identify the mannose-containing receptors on the PMN, the cells were surface labeled with 125I and lysed in 0.5% Nonidet P-40, and the lysate was fractionated by affinity chromatography on a column of Sepharose-bound fimbriae. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the material eluted from the column with 500 mM methyl-alpha-mannoside revealed two radioactive bands of Mr 70,000 to 80,000 (gp70-80) and 100,000 (gp100). Another weak band of Mr 150,000 (gp150) was observed after prolonged exposure of the gel. Upon blotting of the glycoproteins separated by polyacrylamide gel electrophoresis and overlaying of the blots with concanavalin A, gp150 appeared as the major band. Membrane preparations of the PMN were enriched in gp70-80, gp100, and gp150, in comparison with the cell homogenates, further suggesting that these glycoproteins are surface components. Fractionation of the membrane preparations on the immobilized fimbriae followed by concanavalin A overlay of blots of the methyl-alpha-mannoside-eluted material revealed that gp150 was the major component in this fraction. The eluted fraction, obtained from a cell lysate (4.4 micrograms/ml), inhibited by 70% the agglutination of yeasts by the intact bacteria. Our results suggest that the three surface glycoproteins isolated by us serve as receptors for mannose-specific E. coli on PMN and may be involved in the lectin-mediated phagocytosis of the bacteria.     

Purification of merozoites ofTheileria sergenti from infected bovine erythrocytes

A simple and rapid method for purifying merozoites ofTheileria sergenti from infected bovine erythrocytes was developed. Infected erythrocytes were lysed by the use of a cytolytic toxin produced byAeromonas hydrophila and the lysate was subjected to ultracentrifugation in a Percoll discontinuous density gradient. Pure and morphologically intact merozoites free of erythrocyte membrane were recovered from a band formed at the interface of 40% and 60% (by vol.) Percoll solutions. In this merozoite fraction, contamination of erythrocyte membrane proteins was not detected as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Structural components of mouse mammary tumor virus. III. Composition and tryptic peptides of virion polypeptides.

The compositions and possible structural interrelationships among mouse mammary tumor virus (mMTV) proteins were examined following their isolation by preparative sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The minimal carbohydrate content was found to be 9.3% for gp52, 11.2% for gp37.7, and 19.4% for gp33. The carbohydrate moieties of all three glycoproteins were similar in composition, containing fucose, mannose, galactose, N-acetyl glucosamine, and N-acetyl neuraminic acid. One major size class of oligosaccharide side chain was generated from each glycoprotein by pronase digestion of gp52, gp37.7, and gp33. Molecular weights of these glycopeptide size classes were 3200–3600 for gp52, 3750–4250 for gp37.7, and 3200–3700 for gp33. Tryptic peptide patterns of mMTV glycoproteins demonstrated that many of the peptides from gp37.7 and gp33 were similar in size and charge. However, comparison of the tryptic peptides of gp52 with those from gp37.7 and gp33 indicated that gp52 was not structurally related to the other two glycoproteins. SDS-PAGE profiles obtained after limited trypsin digestion of gp52, gp37.7 and gp33 resolved two carbohydrate-containing peptides from each glycoprotein, which appeared to be of similar size. Analyses of tryptic peptides of the five non-glycosylated mMTV proteins, p46, p24, p17, and p8, indicated that these proteins appear to possess distinct amino acid sequences.     

Antigenic cross reactivity between p195 and a distinct protein of 100 kDa in Plasmodium falciparum

A monoclonal antibody raised against merozoites of Plasmodium falciparum clone T9/96 was shown to react with an extremely strain specific epitope on a 195 kDa protein synthesized only by late trophozoites and schizonts. This protein was shown to exhibit all of the characteristics attributed to the molecule known variously as merozoite surface protein precursor, polymorphic schizont antigen and p195. The monoclonal antibody also identified a cross-reactive epitope on a distinct protein of 100 kDa in ring stage parasites which was shown to be synthesized throughout the asexual cycle and was not a processing product of p195. One-dimensional peptide mapping studies suggested that these two proteins share a degree of common sequence or structure.     

Identification of surface membrane proteins and surface membrane antigens of plasmodium chabaudi merozoites

Surface membrane proteins of viable merozoites of Plasmodium chabaudi were iodinated by the Iodogen method and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirteen surface membrane proteins ranging from 22 to 270 kDa were thus identified. Most of these proteins could be immunoprecipitated by sera of mice immunized with extracts of P. chabaudi. A few, however, were precipitated only by sera of mice challenged with living parasites after immunization.     

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.     

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms ofTrypanosoma congolense,T. simiae andT. brucei brucei

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) ofTrypanosoma congolense, T. simiae, andT. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCFT. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38–42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.     

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of 'membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.     

Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA)

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73, P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated, correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activity responsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast, the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing is mediated by at least three distinct proteases.     

Evidence that the major surface proteins of three Leishmania species are structurally related

Promastigotes of Leishmania major LRC-L137, L. donovani LEM 75, and L. tropica LRC-L32 were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of Mr 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps of L. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.     

In vitro development of first- and second-generation schizonts of Eimeria contorta haberkorn, 1971 (coccidia,sporozoa)

Summary Sporozoites of Eimeria contorta inoculated into monolayer cultures of bovine embryonic kidney cells and Madin-Darby bovine kidney cells developed to mature second-generation schizonts, which were first seen 72 hr after inoculation. In cultures of bovine embryonic intestine cells, only mature first-generation schizonts developed. Intracellular sporozoites became U-shaped before transforming into trophozoites with a prominent refractile body. Sporozoite-shaped schizonts were not seen. At 30 hr after inoculation, merozoites began to form as conical protrusions of the surface of schizonts with 25–35 nuclei. Mature first-generation schizonts (25 by 20), with 25–35 merozoites each having two refractile bodies, were first observed at 46 hr. The merozoites averaged 9.5 by 2.0 . After enough sodium taurocholate to make a concentration of 0.01% was added to the cultures, merozoites left their host cells, entered others and transformed into trophozoites within 4 hr. Merozoite formation frequently began with the appearance of conical protrusions at opposite poles of second-generation schizonts. During the late stages of their development, the 8–15 merozoites were spirally arranged around the central residual body. Mature schizonts and merozoites averaged 19 by 8.5 and 22.5 by 1.4 , respectively. In some cultures, larger schizonts with 30–40 merozoites, arranged rectilinearly with respect to the residual body, were observed. On the basis of a comparison with the corresponding stages of E. nieschulzi, it is concluded that E. contorta is a valid species.This investigation was supported by PHS Research Grant No. AI-07488 from the National Institute of Allergy and Infectious Diseases and by a fellowship awarded to B. E. G. Müller by the German National Fellowship Foundation. Published as Journal Paper No. 1339, Utah Agricultural Experiment Station.     

Correlation of tunicamycin-sensitive surface glycoproteins from Trypanosoma cruzi with parasite interiorization into mammalian cells

Trypomastigote forms of Trypanosoma cruzi lose infectivity to cultured mammalian cells when exposed to tunicamycin. Upon reincubation into fresh medium, parasites recover their full penetration capacity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides showed that tunicamycin-treated parasites present several components with altered electrophoretic mobility when compared with controls. Immunoprecipitation with rabbit hyperimmune and human chagasic sera indicated that the surface antigens of approximate molecular masses of 175-180, 120-125, 90-95 and 85 kDa are not encountered in tunicamycin-treated trypomastigotes. By affinity chromatography on wheat germ agglutinin-Sepharose, it was observed that the trypomastigote-specific 85 kDa glycoprotein (Tc-85) is affected by the drug. The other affected components are glycoproteins with affinity for concanavalin A. The results suggest that tunicamycin-sensitive surface glycoproteins from T. cruzi are involved in the parasite interiorization into mammalian cells.     

Two-dimensional gel electrophoretic comparison of proteins from virulent and avirulent strains of Mycoplasma pneumoniae.

The protein composition of the virulent M129 strain of Mycoplasma pneumoniae was compared to that of its homologous avirulent strain by the use of standard one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forty-nine individual M. pneumoniae cell proteins were resolved by this method, and the virulent strain was shown to possess a single high-molecular-weight protein not present in avirulent cells. Variability in the resolution of this particular protein in one-dimensional gels prompted the application of two-dimensional gel electrophoresis to the analysis of M. pneumoniae cell proteins. The sequential use of isoelectric focusing in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension permitted the resolution of a least 142 individual M. pneumoniae cell proteins. Application of nonequilibrium pH gradient electrophoresis in the first dimension achieved the resolution of at least 20 additional basic proteins. Three proteins which are synthesized only by cells of the virulent strain, and not by the homologous avirulent strain, were identified by these two-dimensional gel electrophoresis techniques.     

Identification and characterization of Campylobacter jejuni outer membrane proteins

Outer membrane proteins from isolates of Campylobacter jejuni were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarcosinate-insoluble membrane preparations were outer membrane enriched based on increased ketodeoxyoctonate concentrations, the presence of surface-exposed 125I-labeled proteins that were hydrophobic, and similarity to membrane vesicle (bleb) sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Most isolates contained a single major band with molecular weight of 41,000 to 45,000. Profiles of C. jejuni and Campylobacter coli isolates were indistinguishable, but either could be easily differentiated from Campylobacter fetus and Campylobacter faecalis. The profiles were stable for strains under a variety of growth, incubation and passage conditions. We classified 110 isolates from patients with sporadic campylobacter enteritis into nine subtypes based on differences in outer membrane sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Two categories accounted for 76% of the isolates. Complete concordance was observed in subtypes of strains obtained from epidemiologically related cases. Thus, comparison of the major outer membrane proteins of C. jejuni is a useful technique for investigating the transmission of this organism and may provide a basis for immunological characterization of the outer membrane proteins.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium knowlesi

P. knowlesi parasites with a maximum age distribution of 3 h were metabolically labelled with [³⁵S]methionine during 9 sequential non-overlapping intervals, from young rings to mature segmented schizonts. The proteins synthesised at the different stages were compared using sodium dodecyl sulphate-polyacrylamide gel electrophoresis; more than 40 polypeptides (M_r 20 000 to over 200 000) were identified in the different parasite preparations. The major polypeptides synthesised by rings and trophozoites of different ages were similar, but differences in minor polypeptides could always be recognised. At the onset of schizogony ring and trophozoite specific proteins ceased to be synthesised and proteins specific to schizogony emerged. In general, schizont-specific proteins were of higher molecular weight than ring stage proteins. Details of the morphological changes which occurred during the metabolic labelling episode permits correlation between parasite structure and synthesis of particular polypeptides. Comparison of parasite components metabolically labelled with [³H]glucosamine during defined periods of development also revealed stage-specific synthesis of glycoproteins. The extent to which proteins are altered after synthesis has been investigated by pulse-chase experiments.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium falciparum

Highly synchronised cultures of cloned Plasmodium falciparum (clone T9-94) were metabolically radiolabelled with [35S]methionine during eight consecutive non-overlapping intervals, while parasites developed from young rings to mature schizonts. Analysis of equal amounts of trichloroacetic acid precipitable radioactivity from each interval by sodium dodecyl sulphate-polyacrylamide gel electrophoresis fluorography allowed the stage specificity of protein synthesis to be investigated. More than forty polypeptides with molecular weights of 20 000 to 200 000 can be distinguished. While some proteins are synthesised throughout erythrocytic schizogony many are shown to be stage-specific. Among these are a range of high molecular weight proteins synthesised only during nuclear division. Detailed morphological information permits correlations to be made between synthesis of particular polypeptides and parasite structure.