Eur. J. Biochem. 205,815-825 (1992) $:, FEBS 1992

Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium fakiparum

Angela DIECKMANN-SCHUPPERT ', Stephanie BENDER ', Maria ODENTHAL-SCHNITTLER ', Ernst BAUSE' and Ralph T. SCHWARZ'

' Zentrum fur Hygiene und Medizinische Mikrobiologie, University of Marburg, Federal Republic of Germany lnstitut fur Physiologische Chemic, University of Bonn, Federal Republic of Germany

(Received Dccember 13, 1991/February 7, 1992) - EJB 91 1668

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 intrd- erythrocytic 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 Organiza- tion, 1989). A more thorough understanding of the biochemis- try 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

Correspondence to A. Dieckmann-Schuppert, Zentrum fur Hy- giene und Mcdizinische Mikrobiologie, University of Marburg, Robert-Koch-Str. 17, W-3550 Marburg, Federal Rcpublic of Germany.

Abbreviations. glycosyl-PtdTns, glycosyl-phosphatidylinositol; HPAEC, high pH anion-exchange chromatography; PP, pyrophos- phate; PNGase F, protein N-glycanase F.

Enzymes. Calf intestinal alkaline phosphatase (EC 3.1.3.1); phosphodiesterase (EC 3.1.15.1); protein N-glycanasc F (EC 3.2.2.18); UDP-GlcNAc: dolichol-phosphate GlcNAc-phospho- transferase (EC 2.7.8.1 5).

- --

cycle or glycosyl-phosphatidylinositol (glycosyl-PtdIns) mem- brane-anchor biosynthetic intermediates. Glycoproteins may carry oligosaccharides in N-glycosidic or 0-glycosidic linkage, covalently bound to side chains of the polypeptide, or a glycan may be integrated in glycosyl-PtdIns-anchor structures linked to the C-terminal amino acid. Many glycoproteins are cell- surface proteins or secreted proteins, and the post-trans- lational modification of these proteins by sugars may greatly affect their function, localization and eventual antigenicity.

Few proteins of P. fakiparum have so far been identified as putative glycoproteins (Howard and Reese, 1984; Ramasamy, 1987). The occurrence of terminal galactose was noted as an immunologically important detail (Ramasamy and Reese, 1986). The occurrence of glycosyl-Ptdlns-anchored proteins in P. ,falciparum is suggested both by metabolic labeling (Haldar et al., 1985, 1986; Schwarz et al., 1986, 1987) and by the use of phosphatidylinositol-specific phospholipase C (Braun-Breton et al., 1988, 1990). Inhibitors of protein glycosylation can inhibit the multiplication of P. falciparum in vitro (Udeinya and van Dyke, 1981a, b; Dieckmann- Schuppert et al., 1992a). Taken together, these reports strongly suggest the presence of side-chain-glycosylated pro- teins besides the putative glycosyl-Ptdlns-anchored ones.

816

This study addresses thc question of the occurrence of N- glycosylation and/or O-glycosylation in P . falcipurum by the use of various inhibitors and by radiolabeling combined with the analysis of the biological fate of the radiolabel. We could show conclusively, for the first time, that there is an apparent lack of protein N-glycosylation in the asexual intra- erythrocytic P. fulcipurum, whereas the majority of polypep- tide side-chain glycosylation is of the 0-glycosidic type.

MATERIALS AND METHODS

In vitro culture of P. fakiparum

The P.firlcijiurum strain FCB-1 (identical with FCBR) was obtained from Dr. B. Enders, Behring Co., Marburg, FRG. It was maintained in RPMI 1640 medium supplemented with 25 mM Hepes, 21 mM sodium bicarbonate, 0.37 mM hypoxanthine, 0.1 mg neomycin/ml, and 10% (by vol.) human A + serum (Red Cross Blood Donation Center, Frankfurt/ Main, FRG). The cultures contained human A t erythrocytes (leukocyte-depleted erythrocyte concentrates, Blood Do- nation Center, University of Marburg, FRG) to a hematocrit of up to 5% and were incubated at 37°C in gas-tight containers (modular incubation chamber, Flow) gassed with a mixture of 90% nitrogen/5% oxygen/5% carbon dioxide. Development and multiplication of the cultures were followed by micro- scopic evaluation of Giemsa-stained thin smears. Synchron- ous development was achieved by repeated treatment with 5% sorbitol as described by Lambros and Vanderberg (1979). If not mentioned otherwise, cultures harbouring 28 -40-h-old trophozoites were used for the present study.

Inhibition tests

Inhibition tests were carried out in flat-bottomed micro- titration plates (Falcon). Freshly synchronized cultures of 1 % hematocrit and not more than 1% initial parasitemia (2 f 2- h-old ring stage parasites) were exposed to serial dilutions of the compound to be tested, dissolved in normal culture me- dium. Where other solvents were needed to achieve complete dissolution, appropriate controls without drug were included. After 48 h (if not mentioned otherwise) the multiplication rate was assessed from the number of newly formed ring-stage parasites.

Stage-specific inhibition was investigated by exposing equivalent aliquots of a highly synchronous (-C- 2 h) culture to the respective drug for intervals of 4 h each, followed by two washing steps and reintroduction into culture in drug-free medium (Dieckmann and Jung, 1986). The inhibition was quantitated as above after 48 h.

Metabolic labeling

[8-JH]Guanosine (3.5 Ci/mmol) was obtained from Sigma, all other radioactive substances were from Amersham- Buchler, Braunschweig, FRG. [14C]Uridine (60 Ci/mol) was used at 7.5 pCi/ml, [8-3H]guanosine at 4 pCi/ml, and all other tritiated substances at 100 pCi/ml. Labeling was always performed for 4 h in complete culture medium containing 10% serum. Afterwards, the cultures were washed 3 times in a 10-fold volume of 140 mM NaC1, 2.7 mM KCI, 7.2 mM Na2HP04 and 1.5 mM KH2P04, pH 7.4, followed, if not mentioned otherwise, by lysis of the cells in twice their volume of ice-cold 50 mM Tris/HCI, pH 8.0, 5 mM each of EDTA and EGTA, 1% (massivol.) Nonidet-P40, 1 mM phenyl-

methylsulfonyl fluoride, 5 mM iodoacetamide, 0.1 mM tosyl- lysine chloromethane and 1 pg/ml leupeptin. The lysates were stored at -8O'C. The tritiated bases as well as [3H]ethanolamine (29.5 Ci/mmol) were added directly to the cultures. 3H-labeled fatty acids (myristic, 53.5 Ci/mmol; pal- mitic, 55 Ci/mmol), which are supplied in ethanolic solution, were first dried under nitrogen, then redissolved in a few microlitres of 70% ethanol and added to normal culture me- dium. The final ethanol concentration in the cultures never exceeded 0.02% which, in control experiments, had been pro- ven not to affect the parasites. 3H-labeling with sugar (2- [3H]mannose, 13.6 Ci/mmol; 6-[3H]glucosamine, 25.4 Ci/ mmol;, 6-[3H]galactose, 25.5 Ci/mmol; ~-6-[~H]fucose, 70 Ci/ mmol; l-['4C]mannosamine, 58 Ci/mol) was performed in glucose-free RPMI 1640 medium (Amimed, Muttenz, Switzerland) to which 10 mM fructose had been added (Schwarz et al., 1986). In this case, the cultures were incubated in the glucose-free medium for 30 min before addition of the radioactivity to deplete endogenous glucose stores.

Perchloric acid extraction

Radiolabeled and washed cultures (see above) were di- rectly extracted on ice four times with an equal volume of 0.9 M ice-cold perchloric acid. The extracts were pooled and adjusted to pH 6.5 by the addition of potassium hydroxide. Precipitated potassium perchlorate was removed by centrifugation in a cooled bench-top centrifuge. The extracts were stored at - 80 "C.

Paper chromatography

The analysis of the neutralized perchloric acid extracts was performed by descending paper chromatography on What- man 3MM paper developed in 1 M ammonium acetate pH 3.5 and ethanol ( 2 : 5 , by vol.; Paladini and Leloir, 1952). Standard monosaccharides and derivatives of these were run at 1 pmol/track and visualized by spraying with 1 ?LO aniline in 50% ethanol/O.l M citric acid (Montreuil and Spik, 1968). Radioactivity was detected after cutting the single lanes into 1 cm pieces and counting them in liquid scintillation cocktail (rotiszint eco plus, Roth, Karlsruhe, FRG). Fractions to be further processed were eluted from the corresponding regions with water.

Phosphodiesterase treatment

Samples were dried under nitrogen and redissolved in 40 p1 25 mM Tris/HCl, pH 8.9, containing 0.5 mM magnesium acetate, to which 6 pU enzyme (from Crotulus durissus venom, Boehringer, Mannheim, FRG) were added. Incubation was in a waterbath at 37 'T for 12 h. The reaction products were analyzed by paper chromatography.

Alkaline phosphatase treatment

Samples were processed as for phosphodiesterase treat- ment, except that the buffer contained 10.0 mM magnesium chloride and 0.1 mM zinc chloride. 10 U enzyme (calf intesti- nal alkaline phosphatase, Boehringer, Mannheim, FRG) were added. After 150 min incubation in a waterbath at 37'C, a second aliquot of enzyme was added and the reaction allowed to proceed Tor another 30 min. The reaction products were analyzed by paper chromatography.

81 7

Hydrolysis protocols Monosaccharide constituents of glycoproteins were liber-

ated by hydrolysis in 4 M hydrochloric acid at 1OO"C, for 4 h. Following removal of the acid by methanol evaporation, the monosaccharides were analyzed by high-pH anion-exchange chromatography (HPAEC, see below). To cleave sugars from putative dolichol cycle intermediates, the corresponding frac- tions were subjected to mild acid hydrolysis in 1 M HC1 and 50% 1-propanol at 50°C for 15 min (McDowell and Schwarz, 1988a).

Monosaccharide analysis Monosaccharides were separated and identified by

HPAEC on a CarbopacTM PA1 column (4 mm x 250 mm, Bio- LC, Dionex Co., Sunnyvale, CA) eluted isocratically with 15 mM sodium hydroxide at a flow rate of 1.0 ml/min. Non- radioactive internal standards were detected by pulsed am perometry .

Nucleotide and nucleotide-sugar analysis Putative nucleotide sugars or nucleotides were isolated

by preparative paper chromatography, desalted by charcoal adsorption (Koch et al., 1979) and analyzed by HPLC on a HPTC-AS4A column equipped with a AG4A guard column (Bio-LC, Dionex Co., Sunnyvale, CA) eluted with a gradient over 3-100% 0.5 M NaH2P04, pH 3.4, in water at a flow rate of 1.5 ml/min. Non-radioactive nucleotide and nucleotide sugar internal standards were detected by their ultraviolet absorption at 254 nm.

Organic solvent extraction Glycolipids were extracted from culture lysates with ice-

cold hexane/isopropanol (3:2 by vol., Rosen et al., 1989). To the pooled extracts, one sixth of their volume of water was added. After phase separation, the upper phase was usually devoid of radioactivity or contained the unmetabolized fatty acids, if such were used in the particular experiment. The radioactive lipids in the lower isopropanol/water phase were analyzed by TLC.

TLC analysis Glycolipids were analyzed by TLC on silica G60 plates

(Merck, FRG) developed with chloroform/methanol/acetic acid/water, 25 : 15 : 4: 2 (by vol; Menon et al., 1988). Ethanol- amine and its metabolites were separated on cellulose plates (Merck, FRG) developed with I-butanol/acetic acid/water, 5: 2: 3 (Schneider 1969). The plates were dried and the radioac- tivity distribution analyzed using an automatic TLC-Linear Analyzer (Berthold, LB2842).

Gel filtration This was performed using a Sephadex G25 (Pharmacia)

column measuring 1 cm x 45 cm, equilibrated with 50 mM ammonium acetate. Fractions of 1 ml were collected and the distribution of radioactivity in the eluate monitored by liquid- scintillation counting. Dextran blue (Sigma) served as marker for the void volume.

Protein-N-glycanase (PNGase F) treatment Treatment with PNGase F was preceeded by denaturing

the SDS bound to the protein, 90 pl 20 mM sodium phos- phate, 50 mM EDTA, 10 mM sodium azide and 0.5% (mass/ vol.) Nonidet P40, pH 7.2 were added and the mixture boiled again. PNGase F (Boehringer, Mannheim, FRG) was there- after added at 2 U/ml, and the sample was incubated in a waterbath at 37°C for 24 h. Controls were performed to check the completeness of the PNGase F digestion using hem- agglutinin from influenza A virus labeled with [35S]methionine and in order to ensure the absence of 0-glycan degrading activity in the particular PNGase F batch using bovine sub- maxillary mucin (Sigma).

elimination

The dried sample was taken up in 0.2 M sodium hydroxide and 2 M sodium borohydride and incubated in a sealed tube for 16 h at 45°C. The reaction was terminated by acidification with 50% (by vol.) acetic acid (Montreuil et al., 1986). De- pending on the particular experimental conditions, /3 elimin- ation procedures used to split off 0-glycans may cause exces- sive polypeptide backbone destruction, mimicking the liber- ation of large 0-glycans upon gel-filtration analysis. The con- ditions employed in the present study have therefore been carefully checked using [3sSS]methionine-labeled influenza A hemagglutinin, ascertaining that by unspecific backbone cleavage no fragments smaller than 10 kDa were formed.

Transfer of dolichol-linked oligosaccharides

DolPP(GlcNAc)2, DolPP(GlcNAc)2-Man9, and DolPP- (GlcNAc)2Man9Glc3, (PP, pyrophosphate) labeled with I4C in their glycan moieties, were prepared by a standard pro- cedure (McDowell and Schwarz, 1988 a) using secondary chicken embryo fibroblasts. The purity of these compounds was checked by TLC or Biogel P4 chromatography after acid hydrolysis and endoglucosaminidase H treatment, respective- ly, and found to be > 95%. Oligosaccharyl-transferase activity was determined by incubating 200 - 400 pgprotein (see below) in 50 mM Tris/HCl, pH 7.2, 0.8% (mass/vol.) Triton X-100 and 10 mM manganese chloride, with I O4 cpm of the respec- tive DolPP-oligosaccharide and 1 mM acceptor peptide ( N - henzoyl-Asn-Gly-Thr-NH2) per ml. The reactions were stopped by the addition of methanol and chloroform and the radiolabeled glycopeptides isolated by subsequent phase partition. Dolichol-[ ' 'C]~lig~~accharide~ are recovered in the organic and/or interphase, whereas 4C-glycosyla ted peptides partition into the aqeous phase (Bause et al., 1982). Controls without peptide were performed to check the rate of spon- taneous hydrolysis of the donor oligosaccharide lipid, which was always below 2%.

As the transferase source, we used P. fulcipurum isolated by the saponin method (Goman et al., 1982), subsequently lysed in distilled water containing protease inhibitors for 5 min on ice, then readjusted to the assay buffer conditions. Alterna- tively, microsomes from secondary chicken embryo fibro- blasts or a mixture of both were used.

RESULTS

This study is a first step towards the investigation of glycosylation, in particular the mode of attachment of radiolabeled sugars to P . jalciparum proteins, the nature of this radiolabel and its structure. In contrast to its host cell, the

the sample by boiling for 2 min in 10 p1 1 YO SDS. To displace human erythrocyte, the intracellular parasite P.,falcipanim is

81 8

Fig. 1. Autoradiography of the SDS/polyacrylamide gel electrophoretic analysis of P. fakiparum proteins radiolabeled with [3H[ethanolamine (lane l), 13H(inositol (lane t), (3HJmyristic acid (lane 3), 13Hlglucos- amine (lane 4), 13H]galactose (lane 5), [3H]mannose (lane 6), and I3H]- fucose (lane 7). Molecular mass markers are indicated in kDa.

able to synthesize proteins. P.falciparum-infected cells can be metabolically labeled, not only with amino acids, but also with a variety of sugars, ethanolamine, inositol, and fatty acids (Fig. 1) . Under identical conditions, uninfected erythro- cytes do not incorporate these compounds into proteins (not shown).

As a first approach, the effect on the intraerythrocytic development of P.,fulciparum of some inhibitors of glycosyla- tion, the mode of action of which had been thoroughly studied and defined in other systems (for reviews see Schwarz and Datema, 1982; McDowell and Schwarz, 1988b), was tested. The data obtained are summarized in Table 1. Glucosamine inhibits the multiplication of P. fakiparum at concentrations similar to those needed to inhibit glycosylation in baby ham- ster kidney and secondary chicken embryo fibroblast cells. The same applies for 2-deoxy-~-glucose and the 2-deoxy-~- fluoro sugars, all known to act as mannose analogues in secondary chicken embryo fibroblast cells. Tunicamycin does not exhibit any effect, visible by light microscopy, after 48 h (one developmental cycle) exposure, even at concentrations up to 200 pM. When the exposure time is, however, extended to 96 h (2 cycles), 12 pM tunicamycin is sufficient to kill the parasite. More detailed data on the biochemical effects of tunicamycin on P. ,fulcQurum have been published elsewhere (Dieckmann-Schuppert et al., 1992).

Gel filtration analysis on Sephadex G25 of t3H]sugar- labeled culture lysates revealed that the majority of the radiolabel is recovered in a low-molecular-mass fraction eluting in a broad peak around a relative elution volume of 1.4 (Fig. 2, Al , B1 and Cl), thus far tentatively characterized in the case of galactose-labeling as an anionic, possibly sulfated, 0-glycosidic glycopeptide of around 700 Da, which is sensitive to r-mannosidase, a-galactosidase, p-galactosidase and a-fucosidase (J. Hensel, diploma thesis, University of Marburg, 1991). The peak eluting at the void volume and containing material with a molecular mass greater than 5 kDa, was used as the protein fraction, pooled and an aliquot hereof rechromatographed on the same column (Fig. 2 , A2, B2 and C2). The appearance in this experiment of a fraction of the radioactivity in the included volume upon rechromatography

Table 1. Inhibition of the intraerythrocytic multiplication of P. fakiparum (FCBl) in vifvo by selected inhibitors of glycosylation. The stage affected shows the age of the parasite, related to its 48-h devclop- mental cycle, at which the application of the drug prevents further development. The duration of exposure to the 100% inhibitory con- centration (TCloo) of the respective drug at the most susceptiblt devel- opmental stage is that which prevents further development of the parasite. Note the long exposure time required before bacitracin and GlcN prevent further development of the parasite. Hemolysis of the parasite was obscrved at > 0.4 mM amphomycin. IC50. Concentration causing 50% inhibition; n.d., not determined.

Compound IC50 Stage Exposure affected time for

ICloo until affected

GlcN 25-OH-Cholesterol Compactin Amphomycin Bacitracin Tunicamycin 48 h Tunicamycin 96 h 2-Deoxy-~-glucose 2-Deox y-2-fluoro-

2-Deoxy-2-fluoro-

Castanospermine 1 -Methyl-deoxy-

nojirimycin 1 -Deoxy-

mannojirimycin

glucose

mannose

mM h

4.0 z 0.5 >0.1 >0.4

2.0 > 0.2

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8 -

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n.d. -

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a Decreased twofold in thc presence of 10 mM D-mannOSe.

could be due to some kind of labile radioactivity being present in the untreated sample, which is released by the mock treat- ment involving two boiling steps in the presence of detergent. Another aliquot was PNGase F treated, then rechromato- graphed (Fig. 2, 3A, 3B and 3C). Only in the case of glucosa- mine labeling in five independent experiments did we observe a small (7 - 12%) but reproducible additional shift of radioac- tivity from the void volume towards a relative elution volume of 1.95, indicating enzymatic cleavage. To check the activity of the enzyme and to ensure the absence of endogenous inhibi- tors in P. fakiparum, [35S]methionine-labeled influenza A vi- rus, the hemagglutinin of which is highly N-glycosylated, but not 0-glycosylated, was treated with the enzyme in the pres- ence and absence of a non-radioactive P. fakiparum lysate and run on a SDS/polyacrylamide gel. The enzyme was found to be fully active, resulting in a shift of the hemagglutinin bands towards lower molecular masses, and no inhibition of the enzymatic activity was seen in the presence of the malaria lysate. Two-dimensional gels of normal malarial proteins labeled with [35S]methionine revealed no differences when 10 pg/ml tunicamycin were present during the radiolabeling period (data not shown).

In order to assess the presence of 0-glycans in malaria proteins, lysates from cultures, radiolabeled with tritiated galactose, fucose or glucosamine, were first extensively treated with PNGase F to ensure complete removal of eventual N - glycans, then run on the Sephadex column and the peak

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f r a c t i o n n o Fig. 2. Gel filtration analysis on Sephadcx G25 of P. fakiparum lysates radiolabeled with 13Hjgalactose (A), 13H]fucose (B), or [3Hlglucosamine (C). Lysates were analyzed as a whole (Al , B1, Cl), the material appearing in the void volume hereof rechromatographed after control treatment (no cnzyme added; A2, B2 and C2) or rechromatographed after PNGase F treatment (A3, B3 and C3). Monosaccharides elute at relativc elution volumes 2.9 -3.0.

appearing in the void volume collected as described above. After alkali trcatment, a significant fraction (48 - 63%) of this material was found shifted towards a relative elution volume of 1.9 - 2.2 (Fig. 3), indicating its cleavage from the protein.

Having not detected any N-glycosylated proteins in the asexual intraerythrocytic P . .fulciparum, we therefore then fo- cussed our attention on the search for putative low-molecular- mass precursors for, or intermediates in, protein N-glycosyla- tion to gain more information about the activity of this meta- bolic pathway in malaria parasites. Perchloric acid extracts were prepared from cultures radiolabeled with tritiated man- nose, galactose, glucose, fucose, glucosamine, ethanolamine and ['4C]mannosamine in the absence as well as in the pres- ence of 10 pg/ml tunicamycin and analyzed by paper chromatography (Fig. 4). Ethanolamine had been included in these investigations both for technical reasons and in view of its being a component of glycosyl-PtdIns membrane anchors, which represent another important way of integrating carbo- hydrates into proteins apart from N-glycosylation and 0- glycosylation.

Whereas glucose (Fig. 4C) is, presumably by glycolysis, almost competely degraded, mannosamine (Fig. 4G) is not metabolized at all. In all other panels, peaks in the nucleotide sugar and sugar-phosphate range were found and further

characterized. Radioactive material from single peaks was eluted, treated with phosphodiesterase or alkaline phospha- tase, rechromatographed, and the radiolabeled moiety finally identified by TLC or HPAEC as shown representatively for glucosamine-labeling in Fig. 5. By this procedure, the com- pounds listed in Table 2 could be identified. Glucosamine and ethanolamine are both found present i i s their corresponding nucleotides as well as phosphates, but only in parasitized cultures. Ethanolamine was found to be partially converted to choline. Galactose was partially converted to glucose by parasitized as well as unparasitized cultures. Both these were converted to sugar nucleotides and monophosphates, glucose in addition to the diphosphate. Only a part of the fucose peaks A and B is sensitive to phosphodiesterase or alkaline phosphatase digestion, whereas authentic GDP-fucose and fucose-I -phosphate are fully susceptible. In extracts from mannose-labeled cultures, no nucleotide sugar is found, but most of the radiolabel is recovered as a diphosphate, which disintegrates at pH 8.9 in a two-step manner liberating man- nose.

Radiolabeling with uridine or guanosine yielded a large amount of nucleotide monophosphate, diphosphate and triphosphate, which were selectively removed from the re- spective nucleotide sugars by alkaline phosphatase digestion.

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Fig. 3. Gel filtration analysis on Sephadex G25 of P. falciparum lysates radiolabeled with 13Hjgalactose (lane A), [3Hlfucose (lane B), or ['Hlglucosamine (lane C). Lysates were analyzed as a whole after PNGase F trcatment ( A l , B1 and Cl), the material appearing in the void volume then rcchromatographed (A2, €32 and C2), or rechromatographed after alkaline a-elimination treatment (A3. B3 and C3).

The remaining base-labeled sugar nucleotides coeluted on HPLC with UDP-GlcNAc and with UDP-Gal/UDP-Glc (not separated) in the case of uridine radiolabeling and with GDP- Man/GDP-Fuc (not well separated) in the case of guanosine radiolabeling. The sugar-labeled nucleotide sugars also coeluted with the respective above-mentioned standards, thereby strongly suggesting that galactose and N-acetyl-glu- cosamine are present as UDP derivatives and fucose as the GDP derivative. Again, no radioactivity derived from [3H]mannose eluted at the position of GDP-Man, confirming the results obtained by enzymatic analysis.

When perchloric acid extracts were prepared from cultures radiolabeled with glucosamine in the presence of 10 pg/ml tunicamycin and subsequently analyzed by paper chromdtog- raphy, no difference was seen between the tunicamycin-treated culture extract and the extract from the untreated control. The same applies to radiolabeling with other sugars as well as with ethanolainine (data not shown). The percentages of UDP-GlcNAc, GlcNAcP, GlcNAc and GlcN were 70.3,15.1, 4.6 and 15.1'/0, respectively, in the extract from untrcated culturcs versus 67.0, 16.5, 3.9 and 8.1%, respectively, in that from treated cultures.

In conclusion, the analysis of the perchloric acid extracts showed that galactose, fucose, glucosamine and ethanolamine had been converted to their activated derivatives required for incorporation into protein, whereas the level of GDP- mannose was below the detection limit.

Hydrophobic (glyco)lipids, which could, for example, be dolichol cycle or glycosyl-PtdIns-anchor biosynthetic inter- mediates, were studied by TLC analysis after organic solvent extraction from metabolically labeled cultures (Dieckmann- Schuppert et al., 1992b). N o difference was seen when the cultures were radiolabeled with tritiated glucosamine, palmi- tate, myristate or inositol, in the presence or absence of tu- nicamycin (data not shown). The same holds true for ethanola- mine and myristate radiolabeling and also for an incubation in 10 pg/ml tunicamycin for 12 h prior to radiolabeling (data not shown). All the glucosamine-labeled material resists mild acid hydrolysis under conditions suitable for the cleavage of dolichol-cycle intermediates. Upon the addition of tu- nicamycin to the culture, neither peak disappears nor in- creases.

During protein N-glycosylation, a preformed oligosaccha- ride synthesized as a dolichyl-pyrophosphoryl derivative is

821

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GDP-Nc N*- l -P

1 - - L

-- __ 10 20 30 40 50

distance migrated [ cm]

0 10 20 a0 40 30

distance migrated [cm]

Fig. 4. Paper chromatographic separation of perchloric acid extracts prepared from P. fakiparum cultures labeled with [3Hlmannose (A), 1311jgalactosc (B), 13Hlglucose (C), 13Hlfucose (D), I‘H]glucosamine (E), I’HIethanolarnine (F) or [14C]mannosarnine (C). The positions or corresponding internal standards are indicated above the respective curves. Numbers of peaks refer to Tablc 2. The dashed lines rcpresent the curvcs- obtained using uninfected erythrocytes.

transferred to the nascent polypeptide chain prior to eventu- ally undergoing further modification by trimming reactions. In order to study the transfer reaction, exogenous peptide acceptors mimicking the N-glycosylation site have been suc- cesifully used (reviewed by Kaplan et al., 1987). Microsomes from secondary chicken embryo fibroblasts, which were used as a positive control, readily transferred most of the radioac- tivity and did so, too, when malarial lysate was added, thus ruling out the presence of endogenous transferase inhibitors in the malarial preparation. When, however, a total P . fakiparum lysate alone was used as the enzyme source in such exper- iments, no transferase activity could be detected (Fig. 6), suggesting the absence or inactivity of the enzyme.

DISCUSSION

To date, only a few proteins of the malaria parasite, P . ,fakiparum, have been identified as being metabolically labeled

by sugars and as thus being putative glycoproteins (Howard and Reese, 1984; World Health Organization, 1989). Among these are gp 195, which is the precursor to the major merozoite surface glycoprotein 1 (Holder et al., 1985; Schwarz et al., 1987), and the hrp 2, a secreted histidine-rich protein (Panton et al., 1989). Carbohydrates in general and terminal galactose residues in particular have been implied to confer to the anti- genicity of malarial proteins (Ramasamy and Reese, 1985, 1986). The natural occurrence of antibodies recognizing carbohydrate epitopes on malarial proteins has been reported (Ravindran et al., 1988), but the extent of the contribution of glycoprotein glycan moieties to the antigenicity of malarial antigens is still not clear. In spite of the presence of numerous potential N-glycosybation sites in the P. ,fukipurum proteins listed in the malaria database provided by the World Health Organization and the proven accessibility of the sites in the so-called ‘Serine-rich protein’ to heterologous enzymatic N - glycosylation systems (Ragge et al., 2990) no single malarial

822

1400

- I200

u 1000 g x f: 800 >

0 600

m

Q

o 400

m 200

0

b

0 10 20 30 40 50 0 20 30 40 50

2000 I 1 - - I E P

15001

distance migrated [cm]

distance migrated [cm]

Fig. 5. Enzymatic and HPAEC analysis of the compounds found in the perchloric acid extract obtained from 13HJglucosamine labeled P.Ju/ciparum cultures. Uiitrcated controls are shown in dashed lines. PDE, phosphodiesterase; CIP, calf intestinal alkaline phosphatase.

protein could so far been shown to be N-glycosylated by the parasite. Therefore, this study was aimed at elucidating the inode and extent of protein glycosylation in P. faleiparum.

The ability to radiolabel proteins synthesized in vilro by P . frrlciparuin with sugars and the growth inhibition exerted on P. Ji~lciparum by a number of glycosylation inhibitors in iVrro strongly suggested the presence of sugar-modified pro- teins in this parasite and confirmed earlier observations by Udeinya and van Dyke (1981a, b) concerning the growth- inhibition by 2-deoxyglucose and tunicamycin.

Gel-filtration analyses of sugar-labeled malaria proteins before and after treatment designed to remove N-glycans or 0-glycans specifically, revealed protein 0-glycosylation to be abundant in malarial parasites, 48-63% of the total incor- porated sugar (GlcN, Gal, Fuc) being recovered in 0- glycosidically modificd proteins. Our data on 0-glycosylation in malaria support and extend recent findings by Dayal- Drager et al. (1991), which appeared during the preparation of this manuscript. A fraction (37- 52%) of the sugar-labeled protein was insensitive to our p-elimination procedure. The nature of the chemical linkages in this fraction is under investi- gation.

Very little (GlcN labeling) or no (Gal, Fuc) N-glycosyla- tion of malarial proteins was detectable. The inability of PNGase F to act on malarial proteins had already been de- scribed by others (Ramasamy, 1987). The mere insensitivity of malarial proteins towards PNGase F alone, does, howevcr, not rule out the presence of N-glycans bearing Fuc(ct1-3) at the innermost GlcNAc. which would confer resistance to the enzyme (Tretter et al., 1991). Experiments to elucidate this possibility are currently under way.

Sugars destined to be utilized in protein glycosylation must first be activated by conversion to their nucleotide derivatives prior to eventually being attached to dolichol-(pyro)phos- phate or to being directly incorporated into glycans. Not having found evidence for the occurrence of N-glycosylated proteins in the intraerythrocytic P. jalciparum, the first aspect of glycosylation reactions to be investigated in the malaria parasite was therefore the nature of the hydrophilic sugar derivatives, such as nucleotide and phosphate derivatives formed during metabolic labeling and serving as potential prerequisites for protein N-glycosylation. These compounds can be extracted and simultaneously separated from contami- nating protein by precipitation with perchloric acid. The iden-

Table 2. Identification by enzymatic, HPAEC, and HPLC analysis of compounds in perchloric acid extracts off . fulciparum cultures labeled with different radioactive precursors. The peak number refers to thc numbering in Fig. 4.

Labeled precursor Peak no. Identified as

Mannose 1 Man-bisphosphate 2 Man-monophosphate

Galactose l a Glc-bisphosphate I b UDP-Gal

2 Gal-monophosphate Glc-monophosphate

3 Gal Glc

4 (not identified)

UDP-GIG

Fucose

Glucosamine

Ethanolamine

1 2 3 1 2 3 4 1

2

3

GDP-FUC Fuc-monophosphate Fuc

GlcN Ac-monophosphate GlcN GlcNAc

CDP-Cho EtN-monophosphate Cho-monophosphatc EtN Cho

UDP-GIcNAc

CDP-EtN

Mannosaminc 1 ManN

B c

7-

1

1 '2 3 1 2 :: 1 2 3

Fig.6. Assay of the capacity of a total P. ,falciparum lysate (1) to transfer 1'4C~oligosaccharides from dolichol-pyrophosphoryl derivatives (A, Dol-PP-GlcNAczMan,; B, Dol-PP-GlcNAc2Man9GIc3; C, Dol- PP-GlcNAcz), to the acceptor peptide, N-benzoyl-Asn-Gly-Thr. Con- trols were performed using secondary chicken embryo fibroblast niicrosomes ( 3 ) or a mixture of the malarial lysate and chicken embryo fibroblast microsomes (2). Open bars show radioactivity recovered in the organic phase (material not transferred), black bars represent radioactivity in the aqueous phase (oligosaccharides transferred to the peptide).

tity of the non-radioactive nucleobase in the [3H]sugar-labeled or [3H]ethanolamine-labeled nucleotide derivatives described below is inferred from the results obtained in the uridine-

823

labeling and guanosine-labeling experiments and the elution positions of the respective nucleotide derivatives on HPLC.

The partial conversion of galactose to glucose was not unexpected since the existence of the corresponding epimerase system in erythrocytes has long since been known (Beutler, 1984). Notwithstanding this partial epimerization to glucose, galactose-labeled protein eluting in the void volume (molec- ular mass greater than 5 kDa) upon gel filtration, from which the sugar was subsequently released by acid hydrolysis, exclu- sively contained galactose upon sugar analysis by HPAEC. No glucosylation via galactose was detected. It cannot be decided from the perchloric-acid-extract analyses whether a corresponding galactose/glucose epimerase system is present in P. ,fakiparum and where the respective nucleotide deriva- tives, which should not be membrane permeable, are localized in the infected cell. The fact that the malaria protein contained galactose favors however, the idea of at least some UDP- galactose being formed in the parasite itself.

Of the fucose-labeled peaks 1 and 2 (Fig. 4D), only a part was susceptible to enzymatic degradation by phosphodi- esterase and alkaline phosphatase, respectively. The identity of the insensitive fractions still remains to be elucidated. Fucose- labeled protein was shown to contain only fucose upon HPAEC analysis of the sugar moiety.

The finding that mannosamine was not detected as being incorporated in any metabolite, even when perchloric acid was replaced by 50% aqueous ethanol which in contrast to the acid does not destroy any CMP-neuraminic acid, agrees with an earlier report by Schauer et al. (1984) stating that malaria parasites do not contain or synthesizc sialic acids, the biosyn- thesis of which involves mannosamine.

Mannose was recovered mainly as a diphosphate. This metabolism occurs in parasitized as well as in control erythro- cytes and could therefore be performed by erythrocytic en- zymes. N o radioactive peak was left in the nucleotide-sugar region after alkaline phosphatase treatment, indicating that the level of nucleoside - diphosphate-mannose is beyond the detection limit or that it has not been present at all. Wc did, however, succeed in recovering radioactive mannose from the hydrolysis of an immunoprecipitate of the gp195 from a 2- [3H]mannose-labeled P. fulciparum lysate, suggesting that, nevertheless, some radioactive GDP-Man must have been formed. The lack of detection of GDP-2-[3H]-mannose may explain the extremely low incorporation of radioactive man- nose into malarial proteins (Fig. 1). Glucosamine and etha- nolamine, which both are also structural components of glycosyl-PtdIns membrane anchors, were readily converted to their respective nucleotides and phosphates, accompanied, in the case of glucosamine by N-acetylation, in that of ethanola- mine by methylation of some ethanolamine to choline.

UDP-GlcNAc and CDP-ethanolamine/choline and the corresponding phosphate derivatives were exclusively syn- thesized by the parasite, since the uninfected erythrocytes (Fig. 4) were found devoid of these compounds. This is, to our knowledge, the first report on the occurrence of these glucosamine metabolites, and hence the existence of the corre- sponding enzymes in P. jalciparum. Proteins radiolabeled with glucosamine or ethanolamine, obtained and processed as dc- scribed above, contain unmetabolized glucosamine or etha- nolamine, respectively, indicating that no metabolic conver- sion except for the possible N-acetylation of glucosamine, had taken place during the process of incorporation into proteins. The identity of glucosamine,"-acetylglucosamine is retained, too, in the glycolipid fraction (Dieckmann-Schuppert et al., 1992b). This is in marked contrast to the rapid epimerization

824

of glucosainine to galactosamine and eventually other sugars, which has been observed in liver cells (Warren, 1972) as well as in the parasitic protozoan Toxoplasma gondii (Odenthdl- Schnittler, M., Tomavo, S. and Schwarz, R.T., unpublished results) and suggests that P . ,faleiparum may not possess an active UDP-GlcNAc 4'-epimerase.

The fact that there is no accumulation of UDP-GlcNAc in parasitized erythrocytes in the presence of tunicamycin does not seem compatible with inhibition of UDP-GlcNAc: dolichol-phosphate GlcNAc-phosphotransferase by tunica- mycin, suggesting the enzyme to be inactive or tunicamycin insensitive.

The finding that the pattern of radiolabeled metabolites detected by TLC of organic solvent or paper chromatography of perchloric acid extracts was not changed in response to tunicamycin again suggests the insensitivity of the metabolic pathways concerned. If, by blockage of the dolichol cycle, there was an accumulation not only of UDP-GlcNAc, but also of Dol-P-Man, an increase in the amount of glycosyl- Ptdlns-anchor biosynthetic intermediates as has been ob- served in trypanosomes (Masterson et al., 1989) should have been expected. Apart from this, no dolichol-cycle intermedi- ates were detected among the glucosamine-labeled glycolipids, which is in accordance with corresponding results obtained using a cell-free system (Gerold et al., 1991) and confirms the low activity or inactivity of a putative dolichol cycle in the developmental stage of P. ,faleiparum under study.

The apparent lack of N-glycosylated proteins is in accord- ance with the low or lacking dolichol-cycle activity mentioned above, with the lack of inhibitory activity of tunicamycin against malaria during a single round of multiplication and with the apparent lack of oligosaccharyl-transferase activity, no endogenous inhibitor of this enzyme being detected. Fur- thermore, the multiplication of P . jblciparum in vitro is insensi- tive towards trimming inhibitors such as castanospermine, N - methyl- 1 -deoxy-nojirimycin, 1 -deoxy-mannojirimycin (Table 1). These data may explain the observation by Wright et al. (1991) that no effect on cytoadherence of P . falciparum- infected erythrocytes was seen when the infected cells were treated with the glucosidase and mannosidase inhibitors castanospermine, swainsonine, and deoxymannojirimycin, whereas adherence was readily disrupted upon treatment of the C32 melanoma cells used in the adherence assay. This action of the trimming inhibitors further supports the con- clusion that protein N-glycosylation proceeds, if at all, only to a rather limited extent in the intrderythrocytic trophozoites of P . ,fakiparum. To our knowledge, P. ,falciparum is the first eukaryotic organism reported to be, even if this should be stage-dependent, deficient in N-glycosylation, despite the documented presence of possible N-glycan acceptor sites. N- glycosylation was hitherto found in a number of other para- sitic protozoa, such as different kinetoplastids (Bosch et al., 1988; Olafson et al., 1990; Parodi et al., 1983; Zamze et al., 1990) as well as more closely malaria-related apicompexan T. yondii (Tomavo et al., 1991). Whether genes encoding enzymes participating in protein N-glycosylation can be found in ma- laria parasites, is currently under investigation. We suggest that onc has to be aware of this apparent lack of N-glycosyla- tion in the erythrocytic stage of P . julciparum when expressing recombinant malarial proteins in eukaryotes using vaccinia, baculo virus or a yeast transformation system, all of which, albeit to a different degree, are capable of protein N-glycosyla- tion.

Prof. V. Kretschmer (University Blood Hank, Marburg) kindly provided human erythrocytes for the in iitrv cultivation of P.

.fakipurum. A. Stieneke-Gr6ber (Institute for Virology, Marburg) kindly donated [35S]methionine-labeled influenza A virus [Fowl Plague Virus/Dutch/27 (H7N7), adapted to growth on BHK cells]. This paper includes information obtained from the UNDP/World BankjWHO-TDR malaria sequence database. This study was sup- ported by the Deursche Forschimngsgemein.scI?~~ft, grants no. Schw 2961 4-1 and 29614-2, Fonds der Chwnischen Indirstrie, ARC from British Council/DAAD, Hessisches Ministerium fur Wissenschaft und Kunst, and P. E. Kempkes Foundation, Marburg, FRG.

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