This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium ,fakiparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. fakiparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or 0-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into 0-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. fakiparum, using exogenous glycosyl donors and acceptors.Our studies show that 0-glycosylation is the major form of protein glycosylation in intrderythrocytic P. julciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A,, Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372,6451.Plasmodium fakiparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, today this disease still causes the death of several million people/year (World Health Organization, 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides in P. julcipurum, be they linked to lipids or to proteins. Glycolipids may be membrane components and as such be potential antigens, or be involved in the formation of glycoproteins, e. g. dolichol

The determination of total protein is often a key step for the quantitative analysis of various parameters in tissue and general biochemical research. The classical protocols are restricted to a few compatible buffers, and protocols for the determination of protein in solutions containing protein agglomerates or of protein immobilized on solid surfaces are not available. In such cases, quantification may be complicated. Here, we describe a simple sensitive method for protein quantification circumventing all these restrictions. Proteins in solution or suspension in any buffer are spotted onto cellulose acetate, dried, and stained with Amido Black. After washing off the excess dye, bound Amido Black is solubilized in an acidic solution and determined photometrically. Tissue slices (fixed or native), adherent cell cultures, or Western blots can also be stained and their protein content determined irrespective of the supporting material. A micro-version of the protocol for proteins in solution allows large numbers of samples to be evaluated at a time in microtitration plates and requires only 1-2 microl per sample. A linear concentration dependency (r2=0.950-0.999) was obtained for all samples in all cases investigated. The method presented here permits the exact determination of soluble protein in a large variety of buffers, of insoluble or immobilized protein present on a wide variety of supports, and even of whole cells or tissue slices.

) -EJB 930465/4 0-Glycosylation is the major form of protein glycosylation in human erythrocytes infected with the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum. This study compares aspects of 0-glycosylation in l? falciparum-infected and uninfected erythrocytes.Non-labeled and metabolically glucosamine-labeled 0-glycans were obtained from the protein fraction of infected or uninfected erythrocytes by p elimination. Additional label was introduced by reduction with sodium borohydride, or by the attachment of radioactive Gal to peripheral GlcNAc using galactosyltransferase. 2 -4-times more labeled 0-glycans were obtained from infected erythrocytes compared to the same number of uninfected ones, consistent with additional biosynthesis by the parasite. Our analysis of these 0-glycans showed no significant qualitative divergence between the 0-glycans of the infected and those of the uninfected red cell.According to preliminary alditol analyses, the 0-glycans of P. falciparum-infected red cells do not contain GalNAc at their reducing terminus. Moreover, GalNAc was not synthesized by P. falciparum from either Glc, Gal, GlcN or GalN. At least one 0-glycan found in P. faleiparurn-infected erythrocytes contains GlcNAc at its reducing terminus.Gel-filtration results had suggested the presence of 0-GlcNAc on proteins in the infected erythrocyte. Probing with a synthetic pentapeptide, we could show that P. falciparum expresses its own 0-GlcNAc transferase during intraerythrocytic development. Using this peptide, the enzyme was characterized to some degree. The localization and function of 0-GlcNAc in P. falciparum remains to be elucidated.Plasmodium falciparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, this disease still causes the death of several million people each year. A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Little is known about the biological significance of oligosaccharides in I? falciparum, be they linked to lipids or to proteins.However, post-translational modifications of proteins by the covalent attachment of sugars are often very important for the function, localization and eventual antigenicity of proteins concerned (Rademacher et al., 1988 We have previously shown that glycoproteins of the asexual intraerythrocytic P. fulciparum bear mainly, if not exclusively, 0-glycans, whereas N-glycans seem to be lacking (Dieckmann-Schuppert et al., 1992a). The present study was undertaken in order to investigate the nature of the 0-glycans present in P. faleiparum-infected erythrocytes in general and the occurrence of 0-GlcNAc in particular.Despite the possibility of growing P. falciparum continuously in culture (Trager and Jensen, 1976), the biochemical analysis of this system is hampered by the facts t...

No abstract

No abstract

No abstract

The intracellular protozoan parasite Plasmodium falciparum is the causative agent of malaria in man. The membrane of the merozoites, the invasive stage for the er throcytes, contains, among others, two surface proteins (MSA, anY MSA,), which are presumed to be anchored in the memhrane with a glycosyl-phosphatidyl-inositol (GP1)anchor I l,2,3[. In order to identify and characterize precursorglycolipids f(x GPI-anchors, we have established a cell-free system prepared from P.falciparum trophozoites (33-44 hrs), which are the late erythrocytic stages. Erythrocytes, infected with P.falciparum, were lysed by saponin. The released trophozoites were then lysed by hypotonic shock using a modification of the method described by Masterson et al. 141. Briefly, trophozoites ( 7 . 5~ loy arasites) were resuspended in 6OOpl water containing 0. ImM TECK and Ipglml leupeptin, followed by sonitication (Branson 3200.47kHz) and 30 strokes in a Dounce homogenizer; thereafter an equal volume of IOOmM Na-HEPES (PH 7.4), 50mM KCI. lOmM MgCl , 5mM MnCI,, ,O:ImM TLCK and Ipg/ml leupe tin was added. d i s lysate was divided into aliquots containing 2-5: 108 parasite equivalents. The incubation mixture was completed hy adding ImM ATP, ImM CoA and 2pCi tritiated nucleotide-sugars (UDP-6-[3H]GlcNAc (18.9Ci/mmol, New England Nuclear) or GDP-6-13HIMan (15.1Cilmmol)). Assays were supplemented with I mM GDP-mannose for experiments involving UDP-13HIGlcNAc or with 1mM UDP-GlcNAc for exDeriments HCI, I O O T , 3,3h) of TLC-purified glycolipid V comigrates with a glucosamine standard on High pH Anion Exchange Chromatography (HPAEC, Dionex, Sunnyvale, CA). Three additional more glycolipids, designated glycolipids I, I1 and 111, with R values of Bpd? 0.19, and 0.30, were found in the UDP-[3H]GlcNAc labeled CMextracts. These three gl colipids can also be labeled with GDP-13H]Man. To investigate $e nature of these glycolipids, we have tested their susceptibility to PI-PLC, GPI-PLD, PLA2, HNO , and KOH. The results are summarized in Tahle I. The sensitivity of b e CM-extracted glycolipids to GPI-PLD and nitrous acid treatment share similarities Table 1 Characterisation of Glycolipids synthesized in the cell-free System al CM extract Glycolipldt Rtvalues labeled VIa sensitivity to3 Susceptibility to4 sped.. U-(H)GICNAC G-pHIMan PlPLC PLD PL% H N 4 NaOHI k 0 0 3 + -+ ND II + 0 1 9 + -+ ND 111 + 0 30 + -+ ND I 0.40 + -. ND IV 0 5 5 + ND + + f t 0 5 1 --+ NO V 0 61 + + + + h 012 + + -+ ND VI 1001 7P-Man) 0 81 t B 0 9 1 + -+ ND bl Butanol extpct Glycolipid* Rtvaluas labeled via Sensitivity to G~PHlMan specie. U-IHIGlcNAeC-PHIEtNH4 PlPLc PLD p L b U 0 29 t k -+ t 0 4 1 + + -t + 0 5 1 + d 0.62 + -+ ND -+ ND ! 3 7 + + + + + + NO --+ + + + + + + + + Susceptibility to H N 4 NaOH t t + + + + + +inv~l;ingGDP-[~H)Man and incubated for 45min at 37°C. Glycolipids were extracted twice with Iml chloroform/methanol (CM, 2.1) each, followed hy two extractions with Iml chloroformlmethanol/water (CMW, 1: 1:0.3). The CM-extractable. glycolipids were subjected, to repeated Fol...

The effects of nine different tubulin-binding substances and of one actin-binding compound, cytochalasin B, upon the intraerythrocytic development of the malaria parasite Plasmodium falciparum were investigated in vitro. From the data obtained, plasmodial tubulins seem to be quite different from the mammalian proteins on the molecular level. Tubulozole-T, a substance which is inactive in mammalian systems, appears to be a promising novel antimalarial drug.