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

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<ul><li><p>Eur. J. Biochem. 216,779-788 (1993) 0 FEBS 1993 </p><p>Studies on 0-glycans of Plasmodium-falciparum-infected human erythrocytes Evidence for 0-GlcNAc and 0-GlcNAc-transferase in malaria parasites </p><p>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 </p><p>(Received March 31/June 11, 1993) - EJB 930465/4 </p><p>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. </p><p>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 erythro- cytes 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. </p><p>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. falci- parum from either Glc, Gal, GlcN or GalN. At least one 0-glycan found in P. faleiparurn-infected erythrocytes contains GlcNAc at its reducing terminus. </p><p>Gel-filtration results had suggested the presence of 0-GlcNAc on proteins in the infected eryth- rocyte. 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. </p><p>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 ma- laria biochemistry is the glycobiology of the parasite. Little is known about the biological significance of oligosaccha- rides 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 pro- teins concerned (Rademacher et al., 1988). </p><p>Correspondence to A. Dieckmann-Schuppert, Zentrum fur Hy- giene und Medizinische Mikrobiologie, University of Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany </p><p>Abbreviations. gu, glucose units ; HPAEC, high-pH anion-ex- change 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. </p><p>Enzymes. Calf intestinal alkaline phosphatase (EC; a- galactosidase (EC; P-galactosidase (EC; glucose- 6-phosphate dehydrogenase (EC 1 .I .1.49); bovine milk galacto- syltransferase (EC ; glutamate dehydrogenase (EC; 8-hexosaminidase (EC; phosphodiesterase (EC; protein N-glycanase F (EC </p><p>We have previously shown that glycoproteins of the asex- ual 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. </p><p>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 re- quired to visualize metabolically labeled glycoproteins by autoradiography. To study 0-glycan residues in the P. falei- parum-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 di- rectly 0-glycosidically linked monosaccharide (0-GlcNAc; Hart et al., 1989; Nyame et al., 1987). The modification of </p></li><li><p>780 </p><p>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. </p><p>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 trans- ferase), and we give preliminary data about some biochemi- cal 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. </p><p>MATERIAL AND METHODS </p><p>Parasites and radiolabeling P. falciparum (strain FCBR, asexual intraerythrocytic </p><p>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 radioac- tive 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 un- der culture conditions for 4 days were used as control for the radiolabeling experiments. </p><p>Sample preparation </p><p>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 -80C. Lipids were removed from 50 pl lysate by three sub- sequent extractions with 3 ml ice-cold hexanelisopropanol (3:2, by vol.; Dieckmann-Schuppert et al., 1992b). The resi- due 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 re- move material smaller than 5 kDa (excluded size). The re- maining, delipidated and de-N-glycosylated, protein fraction was used for all further experiments and is hereafter referred to as the 'protein fraction'. </p><p>Galactosylation The equivalent of lo7 cells (uninfected control erythro- </p><p>cytes or red cells from cultures with 10% parasitemia) were treated for 30 rnin at 37 "C with pregalactosylated bovine </p><p>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, essen- tially as described (Torres and Hart, 1984; Holt et al. 1987). Samples to be analyzed by Biogel P4 column chromatogra- phy were then adjusted to pH 9 by the addition of 10 pl 1 M Tris base, and remaining radioactive nucleotide sugar, other- wise interfering with the subsequent chromatographic analy- sis, 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 col- umn o f Dowex AGlX8 (Cl-). </p><p>Release of 0-glycans by reductive p elimination Samples were dissolved in 250 pl 0.05 M NaOHll M so- </p><p>dium borohydride (including, where mentioned, 0.5 mCi NaB[?H],, 73.5 Ci/mmol, Amersham) and incubated at 45C for 16 h. The reaction mixture was neutralized by the addi- tion 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-elimina- tion 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 con- ditions 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). </p><p>Gel filtration </p><p>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). </p><p>High-pH anion-exchange chromatography (HPAEC) </p><p>Saccharide analysis was performed using a BioLC sys- tem (Dionex Co.) equipped with a CxbopakTM PA1 (4 mm X 250 mm) column. Non-radioactive internal stan- dards were detected by pulsed amperometry. For oligosac- charide 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. </p><p>Peptiide glycosylation P: fakiparum parasites, isolated from infected cultures </p><p>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 as- say buffer [final concentrations, if not stated otherwise. </p></li><li><p>781 </p><p>J 10 8 6 5 4 3 2 1 a V V T T T V V V T T </p><p>' "0 n E </p><p>"I 50000 - B E $1 00000 - e </p><p>50000- </p><p>I n </p><p>30 40 50 60 70 80 90 100 110 120 200000 1 </p><p>I r\ </p><p>k </p><p>2 g100000 - </p><p>0 7 50000 - ._ </p><p>e </p><p>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. </p><p>1 I n </p><p>50000 1 0 30 </p><p>25 mM Tris/HCl, pH 7.2, 5 mM MgC12, 5 mM MnCl,, 0.8% (masslvol.) Triton X-1001. The equivalent of 10' P. fulci- purum 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 ra- dioactive 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). </p><p>Enzymic digestions For galactosidase treatment, samples were dissolved in </p><p>50 pl 150 mM sodium phosphatekitrate, pH 6.0, for treat- ment with a-galactosidase, or 50 mM sodium phosphatekit- rate, 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 37C for 48 h, then passed over a mixed-bed ion exchanger </p><p>[Dowex 50WX12 (H') and AGlX8 (formiate form), 0.5 ml each] to remove proteins and salts, and subsequently ana- lyzed 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 37C 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 37C 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. </p><p>Enzyme activity assays Glutamate dehydrogenase (GluDH) was assayed by a </p><p>modification of the proced...</p></li></ul>


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