studies on o-glycans of plasmodium-falciparum-infected human erythrocytes evidence for o-glcnac and...

Eur. J. Biochem. 216,779-788 (1993) 0 FEBS 1993

Studies on 0-glycans of Plasmodium-falciparum-infected human erythrocytes Evidence for 0-GlcNAc and 0-GlcNAc-transferase in malaria parasites

Angela DIECKMANN-SCHUPPERT', Ernst BAUSE' and Ralph T. SCHWARZ' ' Zentrum fur Hygiene und Medizinische Mikrobiologie, University of Marburg, Germany * Institut fur Physiologische Chemie, University of Bonn, Germany

(Received March 31/June 11, 1993) - 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 com- pares 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).

Correspondence to A. Dieckmann-Schuppert, Zentrum fur Hy- giene und Medizinische Mikrobiologie, University of Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany

Abbreviations. gu, glucose units ; HPAEC, high-pH anion-exchange chromatography; V,,, void volume; GlcPDH, glucose-6-phos- phatedehydrogenase; GTF, galactosyltransferase; GluDH, glutamate dehydrogenase; PNGaseF, protein N-glycanase. TosLysCH,Cl, to- syllysine chloromethane ; CIP, calf intestinal alkaline phosphatase.

Enzymes. Calf intestinal alkaline phosphatase (EC 3.1.3.1); a- galactosidase (EC 3.2.1.22); P-galactosidase (EC 3.2.1.23); glucose- 6-phosphate dehydrogenase (EC 1 .I .1.49); bovine milk galactosyltransferase (EC 2.4.1.22) ; glutamate dehydrogenase (EC 1.4.1.13); 8-hexosaminidase (EC 3.2.1.30); phosphodiesterase (EC 3.1.15.1); protein N-glycanase F (EC 3.2.2.18).

We have previously shown that glycoproteins of the asexual intraerythrocytic P. fulciparum bear mainly, if not exclu- sively, 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 continu- ously in culture (Trager and Jensen, 1976), the biochemical analysis of this system is hampered by the facts that only a small percentage of the red cells become infected and that preparations of parasites, isolated using any of the isolation methods available to date, are always contaminated to a vari- able degree by material from the host erythrocytes, which themselves contain 0-glycans. Metabolic labeling of proteins in R falciparum with sugars generally occurs at a very low rate, as indicated by the extremely long exposure times required to visualize metabolically labeled glycoproteins by autoradiography. To study 0-glycan residues in the P. faleiparum-parasitized red cell, this study addressed the total spectrum of proteins in the infected erythrocyte rather than specific ones. Labeling of malarial proteins with radioactive sugars is most efficient with glucosamine. This sugar may not only be an internal or peripheral component of 0-linked oligosaccharides, but is also found in many organisms as directly 0-glycosidically linked monosaccharide (0-GlcNAc; Hart et al., 1989; Nyame et al., 1987). The modification of

780

proteins by 0-GlcNAc seems to be highly dynamic and is supposed to modulate regulatory properties (Haltiwanger et al., 1992a) and may therefore greatly affect the biological activity of the modified proteins.

Based on the results of this study, we provide evidence that the newly synthesized 0-glycans found in P. falciparum- infected red cells bear strong similarities [with respect to their elution behaviour on Biogel P4 chromatography and on high-pH anion-exchange chromatography (HPAEC)] to those found in the uninfected cells. We show for the first time that 0-GlcNAc is formed by malaria parasites and that P. fakiparum synthesizes its own UDP-N-acetylglucosamine : peptide N-acetylglucosaminyl transferase (0-GlcNAc transferase), and we give preliminary data about some biochemical features of this enzyme. Moreover, evidence is provided suggesting that I? fakiparum possesses 0-glycans linked via 0-GlcNAc instead of GalNAc. Some 0-GlcNAc may thus be elongated in malaria parasites.

MATERIAL AND METHODS

Parasites and radiolabeling P. falciparum (strain FCBR, asexual intraerythrocytic

stage) was cultivated in human A ' erythrocytes according to Trager and Jensen (1976), and isolated by isotonic lysis of infected cultures with saponin (Goman et al., 1982). Cultures harbouring 10% parasites in the late trophozoite stage (35- 45 h after invasion) were metabolically labeled with radioactive sugar ([6-'H]GlcN, 25.4 Ci/mmol; [6-'HIGalN, 23.8 Ci/ mmol; [6-'H]GaI, 25.5 Ci/mmol; ~-[6-'H]Fuc, 70 Cilmmol; all from Amersham; [U-I4C]GlcN, 268 mCi/mmol from NEN-Du Pont; tritiated sugars at 0.1 mCi/ml, [I4C]GlcN at 15 pCi/ml) for 4 h in glucose-free RPMI 1640 medium (Amimed). Uninfected erythrocytes which had been kept under culture conditions for 4 days were used as control for the radiolabeling experiments.

Sample preparation

Cultures were washed three times in a 10-fold volume of ice-cold NACl/P, (140 mM NaC1, 2.7 mM KCl, 7.2 mM Na,HPO,, 1.5 mM KH,PO,) followed by lysis of the cells in an equal volume of ice-cold lysis buffer [50 mM Tris/HCl, pH 8.0, 5 mM each of EDTA and EGTA, 1% (masslvol.) Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 0.1 mM tosyllysinechloromethane (TosLys CH,Cl) 1 pg/ml leupeptin]. These lysates were stored at -80°C. Lipids were removed from 50 pl lysate by three subsequent extractions with 3 ml ice-cold hexanelisopropanol (3:2, by vol.; Dieckmann-Schuppert et al., 1992b). The residue was then exhaustively treated with protein N-glycanase, which releases 7-10% of the GlcN label, the nature of which is still unknown (Dieckmann-Schuppert et al., 1992a). Finally, the sample was passed over Sephadex G50 to remove material smaller than 5 kDa (excluded size). The remaining, delipidated and de-N-glycosylated, protein fraction was used for all further experiments and is hereafter referred to as the 'protein fraction'.

Galactosylation The equivalent of lo7 cells (uninfected control erythro-

cytes or red cells from cultures with 10% parasitemia) were treated for 30 rnin at 37 "C with pregalactosylated bovine

milk galactosyltransferase (GTF; Sigma) and 2 pCi UDP-[U- ''C]Gal (325 mCilmmol, Amersham), or 5 pCi UDP-[4, 5- ?H]Giil (39.3 Ci/mmol, NEN-Du Pont), respectively, essentially as described (Torres and Hart, 1984; Holt et al. 1987). Samples to be analyzed by Biogel P4 column chromatography were then adjusted to pH 9 by the addition of 10 pl 1 M Tris base, and remaining radioactive nucleotide sugar, otherwise interfering with the subsequent chromatographic analysis, was degraded by the addition of phosphodiesterase and alkaline phosphatase. When the galactosylation substrate was the glycosylated peptide, the excess of UDP-['4C]Gal was removed by anion-exchange chromatography on a 1-ml column o f Dowex AGlX8 (Cl-).

Release of 0-glycans by reductive p elimination

Samples were dissolved in 250 pl 0.05 M NaOHll M sodium borohydride (including, where mentioned, 0.5 mCi NaB[?H],, 73.5 Ci/mmol, Amersham) and incubated at 45°C for 16 h. The reaction mixture was neutralized by the addition of SO pl 50% acetic acid, followed by repeated methanol evaporation to remove borate as its volatile methyl ester. De- pending on the particular experimental conditions, p-elimination procedures used to remove 0-glycans may cause exces- sive polypeptide backbone destruction mimicking the libera- tion of large 0-glycans upon gel-filtration analysis. The conditions employed here had therefore been optimized in order to ensure that no fragments smaller than 10 kDa were formed by unspecific backbone cleavage (Dieckmann-Schuppert et al., 1992a).

Gel filtration

Size analysis of oligosaccharide alditols was performed using a Biogel P4 column (1 cm X 100 cm) eluted with 0.2 M sodium acetate at a flow rate of 2 ml/h. Each analysis was internally standardized by co-chromatography of partially hydrolyzed dextran, the glucose oliygomers of which were visualized in aliquots of each fraction by the orcinol reaction (White and Kennedy, 1986).

High-pH anion-exchange chromatography (HPAEC)

Saccharide analysis was performed using a BioLC system (Dionex Co.) equipped with a CxbopakTM PA1 (4 mm X 250 mm) column. Non-radioactive internal standards were detected by pulsed amperometry. For oligosaccharide analysis, the column was eluted with a gradient from 0 to 17.5 mM sodium acetate in 100 mM NaOH during SO min at a flow rate of 1 ml/min. Monosaccharides were analyzed under isocratic conditions with 15 mM NaOH at a flow rate of 1 mumin. Fractions of 15 s or 24 s (depending on the particular analysis) were collected, neutralized by the addition of 10 pl 1 M acetic acid, and radioactivity was mea- sured by liquid-scintillation counting.

Peptiide glycosylation P: fakiparum parasites, isolated from infected cultures

with saponin (Goman et al., 1982), were lysed in an equal volume of ice-cold H,O (containing 0.1 mM TosLysCH2C1, 1 pg/ml leupeptin, 1 n M phenylmethyl sulfonyl fluoride, and 5 mhl iodoacetamide) for 5 min, then readjusted to isoos- motic conditions by the addition of double-concentrated assay buffer [final concentrations, if not stated otherwise.

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fractlon no. fmctlon no. Fig. 1. Biogel P4 gel-filtration chromatograms of the oligosaccharide alditols released by p elimination in the presence of NaB['H], from (a) 1.5~10' or (c) 3x10' erythrocytes harbouring 10% l? fakiparum parasitemia, and (b, d) from the respective number of uninfected control erythrocytes. The red cells analyzed in (c) and (d) had been metabolically labeled with [''ClGlcN. The elution positions of hexose oligomer standards are indicated.

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25 mM Tris/HCl, pH 7.2, 5 mM MgC12, 5 mM MnCl,, 0.8% (masslvol.) Triton X-1001. The equivalent of 10' P. fulcipurum was incubated in a total volume of 100 pl of the assay buffer for 30 min. at 37"C, together with lo5 cpm UDP- ['HIGlcNAc and 1 mM of the synthetic acceptor peptide Pro- Tyr-Thr-Val-Val. Control incubations were performed with- out peptide. The reaction was terminated by dilution with 900 p1 ice-cold water and the resulting mixture was passed over a l-ml column of Dowex AGlX8 (C1- form) resin to remove sugar nucleotides and phosphates. The unbound radioactive products were analyzed by chromatography on a Biogel P2 column (1 cmX40 cm, eluted with 0.2 M NH,HCO,), internally standardized by co-chromatography of oligosaccharides of 1 - 10 hexose units (isomaltose oligomers Glc,-Glc,,, from Sigma, plus additional glucose, sucrose, and raffinose) detected by the orcinol reaction (White and Kennedy, 1986). The synthetic peptides Pro-Thr-Val and Pro- Tyr-Thr-Val were used as alternative substrates. Peptides were synthesized as described before (Bause and Legler, 1981).

Enzymic digestions For galactosidase treatment, samples were dissolved in

50 pl 150 mM sodium phosphatekitrate, pH 6.0, for treatment with a-galactosidase, or 50 mM sodium phosphatekitrate, pH 4.5, for treatment with /I-galactosidase, to which 0.5 or 0.125 U of the respective enzyme (u-galactosidase from green coffee beans ; P-galactosidase from jack beans ; both from Sigma) were added. The samples were incubated at 37°C for 48 h, then passed over a mixed-bed ion exchanger

[Dowex 50WX12 (H') and AGlX8 (formiate form), 0.5 ml each] to remove proteins and salts, and subsequently analyzed by HPAEC. For treatment with P-hexosaminidase, samples were dissolved in 100 pl 50 mM sodium citrate, pH 5.0, to which 0.2 U enzyme (from jack beans, Sigma) were added. The samples were incubated at 37°C for 60 h with a second addition of enzyme after 30 h. Phosphodiesterasetreatment was performed in 40 pl 25 mM Tris/HCl, pH 8.9, containing 0.5 mM magnesium acetate, to which 6 pU enzyme (from Crotulus durissus venom, Boeh- ringer) were added. Incubation was in a waterbath at 37°C for 12 h. For alkaline phosphatase treatment, the samples were processed as for phosphodiesterase treatment except that the buffer contained additional 10.0 mM magnesium chloride and 0.1 mM zinc chloride. 10 U enzyme (from calf intestine, Boehringer) 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 for another 30 min.

Enzyme activity assays Glutamate dehydrogenase (GluDH) was assayed by a

modification of the procedure by Schmidt (1970). Briefly, 50 pl samples were incubated at 37°C with 400 pl 132 mM triethanolamine hydrochloride, pH 7.4, 1.2 mM EDTA, 0.16 mM NADPH, and 7 mM 2-oxoglutarate. The reaction was initiated by the addition of 50 pl 1 M ammonium chloride and the decrease in absorbance at 340 nm was followed. Erythrocytic glucose-6-phosphate dehydrogenase (GlcPDH) was assayed by a modification of the protocol by Lohr and

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fraction no. fraction no. Fig. 2. Biogel P4 gel filtration chromatograms of the oligosaccharide alditols released by p elimination in the presence of non- radioactive NaBH, from (a) 5 ~ 1 0 ~ or (c) 5x10' erythrocytes harbouring 10% R fakiparum parasitemia, and (b, d) from the respective number of uninfected control erythrocytes. All cells had been hbeled exogenously with radioactive galactose by bovine milk galactosyltransferase prior to p elimination. Cultures analyzed in (a) and (b) were only galactosylated with UDP-[3H]Gal, whereas the red cells analyzed in (c) and (d) had been metabolically labeled with ['HIGlcN prior to being galactosylated with UDP-['4C]Gal. The elution positions of hexose oligomer standards are indicated. Peaks in (c) are numbered as indicated in the text.

Waller (1970). At 37°C a sample of 20 pl was incubated with 430 pl 167 mM triethanolamine hydrochloride, pH 7.4, 1.5 mM EDTA, 7.6 mM MgCl,, and 0.5 mM NADP. The reaction was initiated by the addition of 50 pl 16 mM glucose 6-phosphate, and the increase in absorbance at 340 nm was followed.

Hydrolysis Monosaccharide moieties of glycoproteins were liberated

by hydrolysis in 4 M hydrochloric acid at 100°C for 4 h. Following removal of the acid by methanol evaporation, the monosaccharides were analyzed by HPAEC.

Re-N-acetylation Samples were re-N-acetylated in 100 mg/ml sodium bi-

carbonate solution by three additions of 2.5% (by vol.) acetic anhydride over 30 min, essentially as described by Ferguson (1992).

Perchloric acid extraction

Labeled and washed cultures (see above) were directly 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. Pre-

cipitated potassium perchlorate was removed by centrifuga- tion 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 Whatman 3 MM paper developed in 1 M ammonium acetate, pH 3.5/ethanol ( 2 : 5 , by vol.; Paladini and Leloir, 1952). Standard monosaccharides and derivatives of these were ana- lysed at l pmol/track and visualized by spraying with l % aniline in 50% ethanol/O.l M citric acid (Montreuil and Spik, 1968 ). Radioactivity was detected after cutting the single lanes into I-cm pieces and analyzing them in liquid-scintillation cocktail (rotiszint eco plus, Roth). Fractions to be further processed were eluted from the corresponding regions with water.

RESULTS The total spectrum of 0-glycans present in P. falcipururn

infected erythrocytes was released from the protein fraction of infected red cell lysates and simultaneously radiolabeled by reductive B elimination in the presence of NaB[3H],. The liberated radioactive oligosaccharide alditols were analyzed

783

by gel filtration on Biogel P4. Selected fractions of the gel filtration eluate were also analyzed by HPAEC. In addition, these /I-elimination experiments were repeated after prior metabolic labeling of the cells with ['4C]GlcN. An identical number of uninfected erythrocytes was always treated in the same manner and served as control. The Biogel P4 radioactivity elution profiles from these two sets of experiments are shown in Fig. 1. They show a major alditol peak eluting near 4 glucose units (gu), flanked by another peak at 2-2.5 gu, and a shoulder at 6-7 gu. All these three major species could also be metabolically labeled by ['4C]glucosamine (Fig. 1 c, d). Although the total amount of 'H- or 14C-cpm recovered after gel filtration varied between four independent experiments; about 2.5 -3 times more 3H-labeled alditols were recovered from infected cells compared to the same number of uninfected red cells (e.g., 2885936 c p d l . 5 X lo7 10% parasitized erythrocytes in Fig. 1 a vs. 1 01 3 357 c p d l .5 X lo' uninfected erythrocytes in Fig. 1 b). In addition, about two- times more [14C]GlcN was each time found metabolically incorporated into infected vs. uninfected erythrocytes (e.g., 4073 cpm in Fig. 1 c vs. 2057 cpm in Fig. 1 d).

To evaluate the occurrence and distribution of terminal GlcNAc in 0-glycans of f? falciparum-infected erythrocytes, the protein fraction of cell lysates was galactosylated via UDP-[3H]Gal by the action of GTF. 0-Glycans were subsequently released by reductive /I elimination, and the liberated radioactive oligosaccharide alditols were analyzed by gel filtration on Biogel P4 (Fig. 2a). Again, uninfected erythrocytes served as control (Fig. 2b). These galactosylation experiments were then repeated using UDP-['4C]Gal after prior metabolic labeling of the culture with ['HIGlcN (Fig. 2c, d). For infected as well as for uninfected erythrocytes, the Bio- gel P4 elution profiles show a non-galactosylated compound (peak 2 in Fig. 2c) eluting at 4 gu, in addition to two galactosylated species eluting at 4.8 gu (peak 1 in Fig. 2c) and close to 2 gu (peak 3 in Fig. 2c), indicating the presence of 0- glycans terminating in GlcNAc and containing about 4 and 1 hexose(s), respectively. In four independent experiments, the total amount of peripheral, GTF-accessible GlcNAc, re- flected by the amount of ['4C]Gal attached to 0-glycans by GTF as calculated from the Biogel P4 profiles, was each time about 30% increased in the protein fraction from infected red cells (e.g., 329059 cpm [I4C]Ga1/5 X lo7 10% parasitized erythrocytes in Fig. 2c vs. 25271 3 cpm [14C]Ga1/5 X lo7 uninfected erythrocytes in Fig. 2d), but the distribution between the different peaks varied. The total amount of ['HIGlcN metabolically incorporated into uninfected erythrocyte material and recovered after Biogel P4 gel filtration (29162 c p d 5 X lo7 erythrocytes in Fig. 2d) was each time about 3 -4-fold lower than that recovered from material corresponding to the same number of P. falciparum-infected cells (105676 cpnd.5 X lo7 erythrocytes harbouring 10% parasitemia, Fig. 2c). Of the galactosylatable peaks, only peak 3 could also be metabolically labeled with glucosamine (Fig. 2c, d). This peak was likely to represent O-glycosidically linked N-acetylglucosamine (0-GlcNAc) and further analyzed in detail. Nothing is known about the identity of peaks 1 and 2 (Fig. 2c, d). Peak 1 should possess terminal GlcNAc, because it can be galactosylated. However, this peak was not labeled with [3H]GlcN during the labeling period and might therefore be of red cell origin. In contrast, peak 2, eluting at about 4 gu, was labeled by ['HIGlcN, but was not galactosylated, indicating that in this case GlcNAc is not the terminal residue. About three-times as much peak 2 material was found in each experiment with f? falciparunz-

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infected vs. uninfected red cells (e.g., 71 092 cpm in Fig. 2c vs. 21 871 cpm in Fig. 2d), suggesting that at least a part of the material may also be of parasitic origin. To assess the identity of the reducing-end sugar(s) of the 0-glycans present in l? fulciparum-infected erythrocytes, cultures were metabolically labeled with tritiated GlcN, Gal, or Fuc. The protein fractions obtained were each divided into two parts, one of which was subjected to reductive p elimination. Both were then separately subjected to complete oligosaccharide hydrolysis. The hydrolysis products were analyzed by HPAEC.

Uninfected erythrocyte cultures did not incorporate ['HIGal or ['HIFuc into proteinaceous material greater than 5 kDa, whereas infected cultures did. From these, Gal and Fuc were always recovered unepimerized, and always in the aldehyde form, irrespective of a preceeding /I elimination, indicating that neither had been present at a reducing-terminal position, in which case the respective alditol should have been formed.

In order to obtain from the ['HlGlcN-labeled material the respective N-acetyl-hexosamine alditols, which in contrast to their respective non-acetylated counterparts are retained by HPAEC, it was necessary to re-N-acetylate the samples prior to HPAEC analysis. Therefore it is not possible to differenti- ate whether the aminosugar had been present in the protein in the acetylated or unacetylated form. However, taking into account that, apart from glycosylphosphatidylinositol structures, all glucosamine integrated into cellular substances known so far is N-acetylated, we may well assume that the material dealt with here had also been N-acetylated. In contrast to Gal and Fuc, [3H]Gl~N was incorporated into material greater than 5 kDa by both infected and uninfected erythrocytes, albeit 3 -4-fold more label (see above) was recovered from infected cells. From uninfected red cells as well as from

7 84

the F! fakiparum-infected material, [3H]GlcN was partially recovered as the corresponding alditol (Fig. 3), if the samples had previously been subjected to reductive P elimination. These results suggest that GlcN(Ac) was incorporated into 0-glycosidic linkages by infected as well as by uninfected erythrocytes during the labeling period. However, from the glucosamine-labeled but not galactosylatable oligosaccharide-alditol, which elutes at 4 gu on Biogel P4 (peak 2 in Fig. 2c and d), GlcNAcitol was recovered, too, but this time only in material from F! falciparum-infected cells (peak 2 in Fig. 2c). This result strongly suggests that Z? falciparum possesses, in addition to 0-GlcNAc, longer 0-glycans closely resembling those of the host erythrocyte, because they co-elute on gel filtration and on HPAEC, but containing GlcN(Ac) at the reducing terminus.

Radiolabeled GalNAc or its corresponding alditol, however, were never detected in Gal-labeled or GlcN-labeled material. Upon metabolic labeling of Z? falciparum-infected cultures with ['HIGalN, no protein material with molecular mass greater than 5 kDa was labeled. The results from a combined paper chromatographic, enzymic, and HPAEC analysis of perchloric acid extracts (performed as described in detail previously, Dieckmann-Schuppert et al., 1992a) from GalN-labeled cells revealed the presence of unmetabo- lized GalN (15.4%) and of GalN-phosphate (84.6%), whereas neither free GalNAc, GlcNAc, nor any N-acetylhexosamine-phosphate or nucleotide sugar could be detected. This reflects the fact which was reported by us before that Z? falciparum is not able to epimerize glucosamine to galactosamine and vice versa, or even to N-acetylate GalN and to form activated compounds thereof.

The double-labeled material (Fig. 2, peak 3) eluting near the position of 2 gu on Biogel P4 (in this P4 system, authentic non-radioactive Gal-GlcNAcitol elutes next to maltose) was pooled and further analyzed by HPAEC. As can be in- ferred from Fig. 4a (not shown separately), more than 97% of the radioactivity in peak 3 eluted on HPAEC with authentic Gal-GlcNAcitol, which had been prepared by alkaline borohydride reduction of commercially available GalPl - 4GlcNAc (Sigma). No prominent impurities were detected.

To further analyze the structure of this presumed disac- charide alditol, the material was treated with P-galactosidase. Upon HPAEC analysis, two radioactive peaks were found, one labeled with 'H and eluting with authentic GlcNAcitol, the other labeled with 14C and eluting with authentic Gal (Fig. 4b). In contrast, no cleavage of the substance was ob- served after treatment with a-galactosidase (Fig. 4a). This proves that peak 3 is Galp-GlcNAcitol, which could only arise from galactosylation of 0-GlcNAc.

The ability of Z? falciparum to form an 0-glycosidic linkage to GlcNAc was assayed in more detail using the synthetic acceptor peptide Pro-Tyr-Thr-Val-Val, containing thre- onine as the potential 0-glycosylation site and lysates from isolated I? falciparum. These transfer GlcNAc from UDP- GlcNAc to this acceptor peptide, forming a radiolabeled compound of about 800 Da (Fig. 5a), which is consistent with the calculated molecular mass of the peptide having one N-acetylhexosamine attached to it (780 Da). From this material, the radioactivity can be released completely as GlcNAcitol by p-elimination (Fig. 5b), indicative of an 0- glycosidic linkage between GlcNAc and the peptide. In uninfected erythrocytes, however, no glycosylation of this peptide via UDP-GlcNAc was detectable under the conditions employed. When F! jalciparum-infected erythrocytes are used as the enzyme source, the activity transferring GlcNAc to

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Fig. 4. HPAEC analysis of the [14C]Gal/[3H]GlcN double-labeled material from peak 3 (Fig.2) (a) after a-galactosidase and (b) after P-galactosidase treatment. The elution profile of the un- treated material was identical to (a). The elution positions of monosaccharides and of alditol standards are indicated.

the peptide increases during the intraerythrocytic growth and multiplication cycle of the parasite (schizogony ; Fig. 6). This increase of 0-GlcNAc-transferase activity parallels that of the specific activity of GluDH, a parasite marker enzyme, which is not present in the uninfected erythrocyte (Vander Jagt let al., 1982). In contrast, the specific activity of the erythrocytic GlcPDH, a marker for red cell mass (Picard- Maureau et al., 1975), decreases during schizogony. Taken together, these data indicate that the activity transferring GlcNAc 0-glycosidically to the peptide is of parasitic origin.

The F! falciparum 0-GlcNAc-transferase was now further characterized. All incubation parameters were as detailed in Material and Methods, only one parameter being varied at a time. The amount of glycosylated peptide formed is linearly correlated with the number of parasites in one assay between 5 X lo6 and 5 X 10'. All further characterisa- tion studies were performed in duplicate with 5 X 10' parasite equivalentdassay. The enzyme exhibits a slightly alkaline pH optimum of pH 8 (Fig. 7a), but shows more than 50% maximal activity in the range between pH 6-9. 0-GlcNAc transfer occurs with a maximal rate at a temperature of 37 "C, but 33% of this activity could still be detected at 24°C. The activity is lost upon incubation at 45°C and hardly measur- able at 5 "C or 15 "C (Fig. 7 b). The formation rate of peptide- 0-GlcNAc is 10-fold stimulated as the divalent metal concentration (magnesium or manganese, respectively) is raised from 0.1 mM to 10 mM (Fig. 7c). The activity is completely abolished in the absence of metal ions or in the presence of equimolar amounts of EDTA. The glycosylated product is stable: at 37°C and pH 7.5 for at least 4 h. The enzyme itself is still active up to this time, as seen by the continuous increase in the amount of product formed (Fig. 7d). How- ever, the addition of lo' cpm fresh UDP-['HIGlcNAc and

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Fig. 5. The synthetic peptide Pro-Tyr-Thr-Val-Val is a potential acceptor substrate for P. falciparum O-GlcNAc transferase. (a) Biogel P2 analysig of the reaction products from the incubation of R ,falciparurn-infected (0) or uninfected (0) erythrocytes with the acceptor peptide Pro-Tyr-Thr-Val-Val and UDP-[3H]GlcNAc. The column was calibrated with hexose oligomer standards, the elution positions of which are indicated. (b) HPAEC analysis of the alditol released by reductive p elimination from the glycosylated peptide shown in (a). Prior to HPAEC analysis, the radioactive material released had been subjected to re-N-acetylation in order to achieve retention of HexNAc-alditols.

retention time [min]

0.5 mM peptide after 2 h does not lead to a further increase in the amount of glycosylated peptide (Fig. 7d), suggesting that enzyme stability rather than substrate availability is the limiting factor. Incubation of P. falciparum lysates with different concentrations of peptide for 1 h each yields saturation kinetics with an apparent K,, of 0.5 mM (Fig. 7e). Synthetic peptides of related sequence but shorter chain length, namely Pro-Thr-Val and Pro-Tyr-Thr-Val-Val, were also recognized by the P. falciparum O-GlcNAc-transferase, but only 10% and 15% of the amount of product formed with the pentapeptide was obtained, respectively (Fig. 7f). To investigate more closely the carbohydrate moiety bound, the glycosylated pentapeptide was treated with P-hexosaminidase. The radioactivity bound to the peptide was completely removed as seen by gel filtration before and after treatment (Fig. 8a), and the liberated radioactive material eluting near 1 gu was identified as GlcNAc by HPAEC (data not shown), suggesting a p-glycosidic linkage between the sugar and the peptide. The GlcNAc-bearing peptide is a substrate for GTF, which, upon incubation with UDP-[14C]Gal, forms a double-labeled substance eluting from the Biogel P2 column slightly ahead of of the ungalactosylated peptide (Fig. 8b). A double-labeled alditol with a molar ratio 'H : 14C of about 1 : 1, eluting at the position of authentic Gal-GlcNAcitol was released

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from the double-labeled substance by reductive P elimination (Fig. 8c), suggesting that only one GlcNAc moiety had been transferred to the peptide by the action of the P. falciparum O-GIcNAc-transferase. The 14C-labeled substance seen in Fig. 8 b and c eluting near 1 gu, is presumed to be galactose or galactitol, the galactose possibly having arisen from UDP- [ I4C]Gal during the galactosylation procedure.

DISCUSSION

Previous work on the glycans of R falciparum-infected erythrocytes had shown the presence of metabolically labeled O-glycans (Dieckmann-Schuppert et al., 1992a; Dayal- Drager et al., 1991 ; Din et al., 1992), in contrast to an apparent lack of N-glycans (Dieckmann-Schuppert et al., 1992a). We now report that infected cells show an increased O-glycosylation potential and also possess O-GlcNAc, which can partially be attributed to a parasite-specific O-GlcNAc-transferase. In addition, we present evidence that O-GlcNAc may be elongated by F! falciparum, whereas classical mucin-type O-GalNAc oligosaccharides are apparently not synthesized.

Upon Biogel P4 gel filtration as well as upon subsequent HPAEC analysis of all the different peaks of the gel-filtration eluates, the radioactivity profiles obtained both after P elimination alone (Fig. l) , and after exo-galactosylation followed by P elimination (Fig. 2), look rather similar when corresponding panels from infected and from uninfected red cells are compared. However, more radioactivity was consistently recovered from the infected culture material, suggesting additional biosynthesis in the parasitized red cells of O-glycosidically linked carbohydrate moieties which are of similar size and possess very similar anionic (HPAEC) properties compared to those structures present already in the uninfected erythrocyte. However, identical elution behaviour on Biogel P4 columns and on HPAEC does not necessarily mean that the underlying structures are completely identical : Oli- gosaccharides in which, for instance, GalNAc is replaced by GlcNAc, might not be resolved by HPAEC. Considering malaria parasite's need to survive within the human host, we may therefore speculate, that molecular mimicry by the syn-

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Fig. 7. Characterization of the F! fulcipurum 0-GlcNAc-transferase. (a) pH dependence (black bars ; 25 mM sodium citrate/phosphate, hatched bars: 25 mM TrislHCl); (b) temperature dependence; (c) effect of Mg2+ and Mn2+ concentration in the incubation buffer; (d) amount of product present after different incubation times (dashed line, addition of new UDP-[3H]GlcNAc and of peptide at 2 h); (e) amounts of product formed in the presence of different concentrations of the peptide substrate; (f) amount of product formed from related shorter peptides. Incubation conditions were as detailed in Material and Methods except for the parameter which was varied. 5 X lox isolated P. fulcipurum were used in each assay and all assays were carried out in dluplicate. The values shown represent the arithmetic mean of these determinations. No activity was ever detected in uninfected erythrocytes.

thesis of similar carbohydrate structures could provide a possibility to escape recognition by the host's immune system.

The results of double-labeling experiments, in which metabolic incorporation of ['HIGlcN into f! fulcipurum-infected erythrocytes was followed by enzymic ['4C]-galactosylation, point to the presence of 0-GlcNAc in the uninfected as well as in the f! fakiparum-infected red cell, because the double-labeled compound Gal-GlcNAcitol was recovered by gel filtration after reductive p elimination. A calculation of the relative amounts of Gal and GlcNAcitol in the disaccha- ride alditol obtained would suggest a 400-500-fold molar excess of Gal over GlcNAcitol, indicating that the specific activity of the ['HIGlcN label was lowered by this factor. This dilution during the metabolic labeling is likely to have

been caused by a large intracellular pool of GlcNAc and/or by a low turnover rate of the labeled proteins. 3-4 times as much ['HIGlcN was incorporated into 0-GlcNAc from parasitized cells (Fig. 2c, d). This result alone, however, does not conclusively prove the synthesis of 0-GlcNAc by the parasite, since the presence of 0-GlcNAc on at least two erythrocytic proteins is well known (Holt et al., 1987) and an activation of the erythrocyte enzyme by the intracellular parasite could so far not be excluded.

The synthetic peptide Pro-Tyr-Thr-Val-Val was shown to become 0-glycosylated by ['HIGlcNAc from UDP- ['HIGlcNAc when lysates of isolated f! fulciparum or of P. fulcil~arum-infected red cells were used as the enzyme source. Despite the documented presence of 0-GlcNAc on

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Fig. 8. Characterization by Biogel P2 chromatography of the carbohydrate moiety attached to the peptide Pro-Tyr-Thr-Val- Val by the II fakiparum 0-GlcNAc transferase. (a) Cleavage of the glycosylated peptide by jack bean P-hexosaminidase; (b) [‘“C]galactosylation of the [’HIGlcNAc-labeled peptide by bovine milk galactosyltransferase (dashed line: elution position of the 3H- labeled peptide before galactosylation) ; (c) release of a double-labeled alditol by reductive p elimination from the double-labeled peptide in (b).

erythrocyte proteins, lysates from uninfected erythrocytes were, in our hands, not capable of transferring radioactivity from UDP-[3H]GlcNAc to this specific acceptor peptide within incubation periods of up to 240 min. This proves un- ambiguously that P. falciparum possesses its own 0-GlcNAc transferase. The erythrocyte enzyme may differ from the red- cell enzyme in terms of acceptor specificity. The particular amino acid sequence of the special peptide used may represent an unsuitable acceptor for the red cell enzyme, or the enzyme modifying the erythrocyte proteins may not be present in the mature red blood cell, whereas its presence was clearly demonstrated in reticulocytes (Starr and Hanover, 1990). The peptide Pro-Tyr-Thr-Val-Val may therefore be used as a specific probe for the P: fakiparum O-GlcNAc- transferase.

The specific activity of the plasmodia1 O-GlcNAc-transferase in lysates of infected red cells increases during growth of the intracellular parasite in parallel to the parasite marker GluDH, indicating that more active enzyme is present in the

mature forms towards the end of the developmental cycle. This is the case for most parasite-encoded proteins and reflects the enhanced activity of protein biosynthesis in those developmental stages.

Haltiwanger et al. (1990) had successfully used another synthetic acceptor peptide, namely Tyr-Ser-Asp-Ser-Pro-Ser- Thr-Ser-Thr, to characterize the activity of the 0-GIcNAc transferase in rabbit reticulocyte membranes. In contrast to that enzyme and to the purified 0-GlcNAc transferase from rat liver (Haltiwanger et al., 1992b), the l? fulciparum iso- form is metal-ion dependent, as is the case for many other glycosyltransferases.

Peptide-bound 0-GlcNAc does not seem to become elongated under the experimental conditions employed, because the size of the alditol, which was obtained by p elimination after galactosylation, only allows for a single GlcNAc residue. This is in contrast to the result mentioned below, that GlcNAc may more generally occur at the reducing terminus of P. falciparum 0-glycans, in places where GalNAc is found in most other eukaryotes. This result may imply that other enzymes, which are not active under the conditions chosen for assaying the malarial 0-GlcNAc-transferase, may be in- volved in the elongation process, or that 0-GlcNAc is bio- synthetically distinct from elongated 0-glycosidically linked GlcNAc, which may substitute GalNAc in malaria.

We conclude that l? falciparum is clearly capable of syn- thesizing 0-GlcNAc, the role of which in malaria remains to elucidated. We do not know which proteins receive 0- GlcNAc by the malarial transferase. They would be assumed to be parasite-encoded proteins, but as long as data on single proteins are not available, transfer to red cell proteins cannot be excluded. In this context, it is interesting to note that data recently published by Din et al. (1992) give evidence for the presence of GlcNAc in 0-glycans of yet undefined molecular structure on the P: falciparum merozoite surface protein p195.

GalNAc, which is normally present at the reducing terminus of mucin-type 0-glycans and is formed from GlcNAc by enzymic epimerization, has so far not been detected in malarial proteins (Dieckmann-Schuppert et al., 1992a). Neither are GlcN or Gal metabolized to GalN(Ac) by asexual intraerythrocytic P: fulciparum (Dieckmann-Schuppert et al., 1992a). The existence of a metabolic source for GalNAc in I? falcipurum has therefore to be questioned. The occurrence in human serum of galactosamine, which could serve as an alternative source of GalNAc for the malaria parasite, has to our knowledge never been reported. We did not succeed i n metabolically labeling infected erythrocytes with GalN. Ex- ogenous GalN, if present in the serum at all, is therefore unlikely to represent a source of GalNAc for l? falciparum. To investigate the nature of the innermost (i.e., reducing-end) sugar(s) of 0-glycans in P. fakiparum-infected red blood cells, alditol analyses were performed on metabolically labeled protein fractions. These revealed the presence of GlcNAc, but not of Gal or Fuc, at the reducing terminal position of 0-glycans in the P: fatciparctm-infected red cell. After complete hydrolysis of oligosaccharide-alditols labeled with various radioactive sugars, only GlcN was, after re-N-acetylation, detected by HPAEC as the corresponding alditol.

In addition to the presence of 0-GlcNAc as such on proteins of the infected red cell, we could also demonstrate the presence of GlcNAc at the reducing terminus of a longer 0-glycosidically linked oligosaccharide from P. fulciparum- infected erythrocytes (peak 2, F i g . 2 ~ ) . In contrast to this, Haltiwanger et al. (1992a) could not detect elongation of 0-

GlcNAc attached to a synthetic acceptor peptide by micro- somes from murine lymphoma cells, and conclude that 0- GlcNAc does not become elongated in those cells. Our results therefore suggest that the malaria parasite can elongate 0-glycosidically linked GlcNAc to form longer 0-glycosidic structures and that l? fakiparum, as opposed to higher eukaryotes, can substitute GlcNAc for GalNAc at the reducing terminus of 0-glycans.

R falciparum strain FCBR was obtained from Dr B. Enders, Behring Co., Marburg, Germany. Prof. V. Kretschmer (University Blood Bank, Marburg) kindly provided human erythrocytes for the in vitro cultivation of P. fakiparum. The authors thank Stephanie Koslowski and Sabine Kauer for their skillful technical help. This work was supported by grants from the Deutsche Forschungsgem- einschaft to R. T. S. (Schw 29614-2 and 29614-3) and to E. B. (SFB 284), from the Fonds der Chemischen Industrie to R. T. S. and to E. B., from British CouncilDAAD (ARC), from the Hessisches Ministerium fur Wissenschaji und Kunst, and from P. E. Kempkes Foundation, Marburg, Germany.

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studies on o-glycans of plasmodium-falciparum-infected human erythrocytes evidence for o-glcnac and...

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