Eur J Biochem 271, 2204–2214 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04150.x Glycosphingolipids in Plasmodium falciparum Presence of an active glucosylceramide synthase Alicia S Couto1, Carolina Caffaro1, M Laura Uhrig1, Emilia Kimura2, Valnice J Peres2, Emilio F Merino2, Alejandro M Katzin2, Masae Nishioka3, Hiroshi Nonami3 and Rosa Erra-Balsells1 CIHIDECAR, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; 2Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Brazil; 3College of Agriculture, Ehime University, Matsuyama, Japan Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite Two different experiments, using UDP-[14C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide The enzyme activity could be inhibited in vitro with low concentrations of D,L-threo-phenyl-2-palmitoylamino3-morpholino-1-propanol (PPMP) In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [14C]palmitic acid and [14C]glucose in the three intraerythrocytic stages of the parasite The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UVMALDI-TOF mass spectrometry When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy Malaria is the most serious and widespread parasitic disease in humans Each year, approximately 300 million people become infected and 2–3 million people die as a result In addition there is considerable morbidity associated with this disease [1] The glycobiology of Plasmodium falciparum has been causing an increasing amount of interest in recent years The presence of N-linked glycoproteins in relation to schizogony of the intraerythocytic stages [2] and glycosylphosphatidylinositols as the major carbohydrate protein modification have been described in the human malaria parasite [3–6] In addition, lipid metabolism has also been attracting a lot of attention with respect to basic biology and applications for malarial chemoterapeutic purposes [7] However, little is known about glycosphingolipids (GSLs), a group of ceramide-based lipids that in other systems regulate interactions of the cell with its environment and play a role in cell signalling [8,9] The first evidence of the presence of GSLs in P falciparum was obtained by metabolic incorporation of [3H]serine and [3H]glucosamine After labeling with the carbohydrate precursor, hydrophilic glycosphingolipids migrating slower than the penta-glycosylated ceramide standard were detected [10] More recently, the synthesis of chloroplast galactolipids in apicomplexan parasites was reported [11] Biosynthesis of complex GSLs in mammalian cells involves sequential glycosyltransferase reactions, starting with the formation of glucosylceramide (GlcCer), and it has been assumed that the various transferases used are functionally organized within the Golgi [12,13] It is known that the key step involves the transfer of glucose to ceramide from UDP-glucose, catalyzed by the action of a glucosylceramide transferase [EC 2.4.1.80: glucosylceramide synthase (GCS)] With regards to localization, as far as it is known, GlcCer is special because it is the only glycosphingolipid synthesized on the cytosolic leaflet in the early Golgi but it is used for the synthesis of higher sphingolipids Correspondence to A S Couto, CIHIDECAR, Departamento de ´ ´ Quı´ mica Organica, Pabellon II, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, 1428, Argentina Fax/Tel.: + 54 11 4576 3346, E-mail: acouto@qo.fcen.uba.ar Abbreviations: GSLs, glycosphingolipids; GlcCer, glucosylceramide; GCS, glucosylceramide synthase; BODIPY-DHCer, BODIPYdihydroceramide; BODIPY-Cer, BODIPY-ceramide; D,L-threo-PPMP, D,L-threo-phenyl-2-palmitoylamino-3-morpholino1-propanol, d18:0, 4-hydroxysphinganine; d20:0, 4-hydroxyicosasphinganine; C10:0, etc., decanoic acid, etc.; C10h:0, etc., hydroxydecanoic acid; C10-2h:0, dihydroxydecanoic acid; C10-3h:0, trihydroxydecanoic acid, etc Enzyme: glucosylceramide synthase (EC 2.4.1.80) (Received December 2003, revised 26 February 2004, accepted April 2004) Keywords: dihydroceramide; glucosylceramide synthase; glycosphingolipids; malaria; Plasmodium falciparum Ó FEBS 2004 Glycosphingolipids in Plasmodium falciparum (Eur J Biochem 271) 2205 in the lumenal leaflet [14] Because glucosylceramide is a pivotal precursor of numerous GSLs, this enzyme is extremely important for understanding GSL function In this report, we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of P falciparum Two different experiments, using UDP-[14C]glucose as donor or fluorescent ceramides as acceptors were performed In both cases, the enzyme showed specificity for dihydroceramide as substrate The enzyme activity could be inhibited in vitro with D,L-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) In addition, GSLs were shown by metabolic incorporation of [14C]palmitic acid and [14C]glucose in the three intraerythrocytic stages of the parasite UV-MALDI-TOF mass spectrometry proved that four fractions analysed corresponded to ceramides, monohexosylceramides, trihexosylceramides and tetrahexosylceramides, respectively When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of GSL biosynthesis was shown The particular substrate specificity of the malarial GCS suggests that this enzyme might represent a new attractive target for malarial chemotherapy Materials and methods Materials Lipid standards and BSA were purchased from Sigma AlbuMax IÒ was obtained from Gibco BRL Life Technologies (New York, NY, USA) All solvents were of analytical or HPLC grade PercollÒ was purchased from Pharmacia Chemicals (Uppsala, Sweden) D,L-threo-phenyl2-palmitoylamino-3-morpholino-1-propanol (PPMP) was from Matreya (Pleasant Gap, PA, USA) and ceramide glycanase from GlyKo, BODIPYÒ-sphingolipids used were from Molecular Probes Polyclonal antibodies against human GCS were a kind gift of D L Marks and R E Pagano, Mayo Clinic Foundation, Rochester, MN, USA TLC was performed on silica gel 60 precoated plates (Merck) using the following solvent systems: (a) chloroform/methanol/water (65 : 25 : 3, v/v/v); (b) chloroform/ methanol/0.25% KCl (80 : 30 : 2, v/v/v); (b), chloroform/ methanol/1 M NH4OH (40 : 10 : 1, v/v/v); (d) chloroform/methanol/water (65 : 25 : 3, v/v/v); (e) chloroform/ methanol/water (80 : 20 : 2, v/v/v) In all cases, radioactive samples were located by fluorography at )70 °C using EN3HANCE (NEN) and Kodak X-OMAT AR films (Amersham, 291 mCiỈmmol)1, 1,54 mCiỈmg ) was incorporated at a concentration of 6.25 lCiỈmL)1 in RPMI 1640 medium without addition of 11 mM of glucose Parasites (5.9% ring forms, 5.4% trophozoites, 3.7% schizonts) were labeled for 18 h 14 )1 D-[U- C]galactose (Amersham, 306 mCiỈmmol , 1,61 )1 mCiỈmg ) was incorporated at a concentration of 3.2 lCiỈmL)1 in RPMI 1640 medium without addition of 11 mM of glucose Parasites (9.8% ring forms, 3.0% trophozoites, 1.6% schizonts) were labeled for 18 h The viability of the parasites was verified by microscopic evaluation of Giemsa stained smears Each stage was purified on a 40/70/80% (w/v) discontinuous Percoll gradient (15 000 g, 30 min, 25 °C) This procedure yielded an upper band (40%) containing schizonts, another band with trophozoites (70–80% interface) and a pellet of ring forms [2] A control containing a similar number of uninfected erythrocytes was incubated with the different radioactive precursors and further processed under the same conditions In order to evaluate biosynthesis of proteins, synchronous P falciparum ring-stage cultures, with a parasitemia of around 5%, untreated or treated with lM PPMP for 48 h, were labeled with 25 lCiỈmL)1 of L-[35S]methionine (> 1000 CiỈmmol)1) (Amersham) in 10 lM methioninedeficient RPMI medium, at the beginning or after 24 h of treatment Aliquots were collected at different times (0–48 h), precipitated with 12% (w/v) trichloroacetic acid, and radioactivity was measured with a Beckman 5000 b-counter 14 D-[U- C]glucose )1 Treatment of parasites with D,L-threo-phenyl-2palmitoylamino-3-morpholino-1-propanol Parasite cultures (6.4% ring forms, 2.4% trophozoites, 1.2% schizonts) were incubated with lM D,L-threophenyl-2-palmitoylamino-3-morpholino-1-propanol (D,Lthreo-PPMP) After 24 h of treatment, parasites were labeled with [14C]palmitic acid or [14C]glucose for 18 h in the presence of the drug After the labeling period, each stage was purified on a PercollÒ gradient as described above and freeze-dried prior to lipid extraction The effect on parasite development was monitored by microscopy of Giemsa-stained blood smears in two independent experiments In all cases, control cultures without the inhibitor and a similar amount of uninfected erythrocytes were labeled under the same conditions Cultures of P falciparum and metabolic labeling Isolation and purification of glycosphingolipids An isolate (S20) of P falciparum obtained from a patient living in Porto Velho (Rondonia, Brazil) was used [15] ˆ Parasite cultures of P falciparum were performed as described [2] [U-14C]Palmitic acid (Amersham 822 mCiỈmmol)1, 2.91 mCiỈmg)1) originally supplied in toluene was dried under nitrogen, redissolved in ethanol, and coupled with defatted BSA at a : (v/v) molar ratio The final labeling medium contained 6.25 lCiỈmL)1 of the radioactive precursor, 0.5% (w/v) Albumax and 0.05% (w/v) BSA Parasites were labeled for 18 h Each intraerythrocytic stage of P falciparum was extracted with chloroform/methanol : (3 · mL) Each extract was fractionated by anionic exchange chromatography on a DEAE-Sephadex A-25 (acetate form) column, which was eluted with chloroform/methanol/water (30 : 60 : 8, v/v/v) to recover neutral GSLs and zwitterionic lipids Anionic lipids were bulk eluted with chloroform/methanol/0.8 M NaAcO (30 : 60 : 8, v/v/v) The unbound fraction was evaporated to dryness and treated with 0.1 M NaOH in methanol (500 lL), at 37 °C for h The mixture was neutralized with HCl M in the presence of M phosphate Ó FEBS 2004 2206 A S Couto et al (Eur J Biochem 271) buffer pH (50 lL) to avoid over-acidification After evaporation, salts were removed by reverse-phase chromatography, using a Sep-Pack C-18 cartridge (Worldwire Monitoring, Horsham, PA, USA) Acidic lipids were also concentrated through a Sep-Pack cartridge Purification of neutral glycosphingolipids was achieved by chromatography on silicic acid The sample was dissolved in chloroform and loaded into a column of Unisil (7 · 50 mm) which was eluted with chloroform (20 mL), chloroform/methanol (98 : 2, v/v, 20 mL) and chloroform/methanol (1 : 3, v/v, 25 mL) [16] In another experiment, total lipids from schizont forms were extracted and purified as described above The purified neutral GSL fraction was analysed in parallel with an analogous fraction obtained from [U-14C]palmitic acid labeled parasites by TLC in solvent B Spots corresponding to the ceramide fraction (I), monohexosylceramide fraction (II), trihexosylceramide fraction (IV) and tetrahexosylceramide fraction (V) were extracted from the plate and analysed by UV-MALDI-TOF MS Acid methanolysis and methylation The sample was hydrolysed for 18 h at 80 °C with 12 M HCl/MeOH/water (3 : 29 : 4, v/v/v) The hydrolysate was dried and the acid eliminated by several evaporations with addition of water Methylation of fatty acids was carried out with BF3/MeOH in dry toluene under nitrogen at 80 °C for 90 [17] Ceramide glycanase digestion Samples were dissolved in 250 mM phosphate buffer pH 5.0 (100 lL) containing 1% (w/v) sodium cholate Ceramide glycanase (from Macrobdella decora) (0.3 mU) was added and digestion was performed at 37 °C for 18 h Lipids were extracted with chloroform/methanol (1 : 1, v/v) and analysed by TLC Glucosylceramide synthase assay Parasite homogenates were prepared in 0.1 M sodium phosphate buffer (pH 7.4) containing mM MgCl2, 25 mM KCl, mM phenylmethanesulfonyl fluoride, mM N-a-tosyl-L-lisine chloromethyl ketone hydrochloride (TLCK) and 10 lgỈmL)1 leupeptine by probe sonication three times with 10 pulses while on ice Liposomal substrate was performed with dipalmitoylphosphatidylcholine and ceramide (palmitoyldihydrosphingosine or palmitoylsphingosine) (10 : 1, v/v) containing 0.1 nmol of ceramide The constituent lipids were dissolved in chloroform/methanol (1 : 1, v/v), vortexed and dried under nitrogen Lipids were dispersed in 0.1 M sodium phosphate buffer pH 7.4 by sonication at °C The reaction mixture consisted of UDP-[14C]glucose (1 lCi, 319 mCiỈmmol)1, Amersham), mM b-NAD and the liposomal substrate (600 nmol lipid phosphorous) in 0.1 M sodium phosphate buffer (pH 7.4) The cell homogenate (50–100 lg protein per tube) was added making a total volume of 15 lL The mixture was incubated at 37 °C for h with shaking Incubations were stopped by freezing and the mixtures were cleaned by passage through C18 cartridges Lipids were eluted with chloroform/methanol (1 : 1, v/v) and further analysed by TLC When the inhibition test was performed, PPMP (5 lM) was added to the reaction mixture Spots were quantified using a phosphoimager (Molecular Analyst, Bio-Rad) with MOLECULAR ANALYST software In another experiment, BODIPY analogues of ceramides were used BODIPY-dihydroceramide (BODIPY-DHCer) was synthesized from dihydrosphingosine and BODIPY acid according to Kok & Hoekstra [18] The enzyme assay was performed in tubes precoated with dipalmitoylphosphatidylcholine (15 nmoles added in chloroform and dried down under nitrogen) by adding the fluorescent ceramide (300 ng per tube) precoupled to BSA, the parasite lysates (50–100 lg protein) and the assay buffer (0.1 M sodium phosphate buffer pH 7.4 containing 25 mM KCl, mM MgCl2, 2.5 mM UDP-glucose and mM b-NAD) in a final volume of 150 lL The reaction was incubated at 37 °C for h with shaking The mixture was extracted with chloroform/methanol (1 : 1, v/v) and analysed by TLC Spots were visualized using a Fuji LAS1000 densitometer equipped with IMAGE GAUGE 3.122, software, Fuji Film, Japan All protein determinations were performed using Bradford’s method [19] Immunoprecipitation Parasite lysates (1–2 mg protein) were incubated with GCS 1.2 antibody (which recognizes a region near the GCS C-terminus) [20] in buffer Tris/HCl pH 8.0 containing 150 mM NaCl, 0.5% (w/v) sodium deoxycholate and 0.1% (w/v) SDS, for h at °C Protein A-Sepharose (10% in the same buffer, 100 lL) was added and it was incubated for a further 60 The mixture was centrifuged at 10 000 g and the immunoprecipitate was washed (3 · 100 lL) The immunoprecipitates were dissolved in sample buffer and subjected to SDS/PAGE in 10% gels Western blot to poly(vinylidene difluoride) membrane was performed and blots were probed with anti-peptide polyclonal antibodies GS-5.1 (1/1500) which recognizes a region near the GCS N-terminus [20] followed by an anti-rabbit horseradish peroxidase secondary antibody and visualized using ECLÒ (Amersham) enhanced chemiluminescence reagent UV-MALDI-TOF MS analysis Matrices for UV-MALDI-TOF MS The b-carboline (9H-pyrido[3,4-b]indole), nor-harmane and 2,5-dihydroxybenzoic acid were obtained from Aldrich Chemical Co Calibrating chemicals for UV-MALDI-TOF analysis a-Cyclodextrin (cyclohexaamylose, Mr 972.9), b-cyclodextrin (cycloheptaamylose, Mr 1135.0), c-cyclodextrin (cyclooctaamylose, Mr 1297.1), angiotensin I (Mr 1296.49), neurotensin (Mr 1672.96) and bovine insulin (Mr 5733.5) were purchased from Sigma-Aldrich Solvents Methanol, ethanol, acetonitrile (Sigma-Aldrich HPLC grade) and trifluoroacetic acid (Merck) were used as purchased without further purification Water of very low conductivity (Milli Q grade; 56–59 nSỈcm)1 with PURIC-S (ORUGANO Co., Ltd, Tokyo, Japan) was used Ó FEBS 2004 Glycosphingolipids in Plasmodium falciparum (Eur J Biochem 271) 2207 UV-MALDI-TOF-MS experiments Measurements were performed using a Shimadzu Kratos, Kompact MALDI (pulsed extraction) laser-desorption time-of-flight mass spectrometer (Shimadzu, Kyoto, Japan) equipped with a pulsed nitrogen laser (kem ¼ 337 nm; pulse width ¼ ns), tunable pulse delay extraction (PDE), post source decay (PSD) (MS/MS device) and a secondary electron multiplier Experiments were first performed using the full range setting for laser firing position in order to select the optimal position for data collection, and secondly fixing the laser firing position in the sample sweet spots The samples were irradiated just above the threshold laser power for obtaining molecular ions and with higher laser power for studying cluster formation Thus, the irradiation used for producing a mass spectrum was analyte-dependent with an acceleration voltage of 20 kV Usually 50 spectra were accumulated All samples were measured in the linear and the reflectron modes, in both the positive- and the negative-ion mode The stainless steel polished surface sample-slides were purchased from Shimadzu Co., Japan (P/N 670-19109-01) Polished surface slides were used in order to get better images for morphological analysis with a stereoscopic microscope (NIKON Optiphot, Tokyo, Japan; magnification ·400) and with a high-resolution digital microscope (Keyence VH-6300, Osaka, Japan; magnification ·800) Sample preparation Matrix stock solutions were made by dissolving mg of the selected compound in 0.5 mL of : (v/v) methanol/water Analyte solutions were freshly prepared by dissolving the samples (0.05 mg) in chloroform/ methanol, : (v/v) (0.025 mL) To prepare the analyte-matrix deposits two methods were used Method A; thin-film layer method (sandwich method) Typically 0.5 lL of the matrix solution was placed on the sample probe tip, and the solvent removed by blowing air at room temperature Subsequently, 0.5 lL of the analyte solution was placed on the same probe tip covering the matrix and partially dissolving it, and the solvent was removed by blowing air Then, two additional portions (0.5 · lL) of the matrix solution were deposited on the same sample probe tip, producing a partial dissolution of the previously deposited thin-film matrix and analyte layers The matrix to analyte ratio was : (v/v) and the matrix and analyte solution loading sequence was: (a) matrix, (b) analyte, (c) matrix and (d) matrix Method B; mixture method The analyte stock solution was mixed with the matrix solution in : 1–1 : 12 (v/v) ratio A 0.5 lL aliquot of this analyte-matrix solution was deposited onto the stainless steel probe tip and dried with a stream of forced room temperature air Then, an additional portion of 0.5 lL was applied to the dried solid layer on the probe, causing it to redissolve partially, and the solvent was removed by blowing air The resulting solid partially crystalline layers were found to be relatively homogeneous in both cases norHarmane and 2,5-dihydroxybenzoic acid as matrices showed signals of higher quality by using Method A Thus, the results shown and discussed in the present article are those obtained using this sample preparation method for each analyte, in the optimum experimental conditions Spectra were calibrated using external calibration reagents: (a) commercial proteins (neurotensin; angiotensin I; bovine insulin) and (b) a-, b- and c-cyclodextrins with norharmane as matrix, in positive- and in negative-ion mode The KRATOS KOMPACT calibration program was used Results Metabolic labeling of GSLs Cultures of P falciparum with parasitemia around 10% (4.5% ring forms, 2.7% trophozoites and 1.6% schizonts) were metabolically labeled with [14C]palmitic acid for 18 h The different stages were purified on a Percoll gradient and extracted with chloroform/methanol (1 : 1, v/v) A control of uninfected erythrocytes was analysed in parallel (Table 1) The different extracts were further fractionated by DEAE-Sephadex A-25 (ACO–) column chromatography into neutral and acidic lipids TLC analysis of the unbound fraction showed that the radioactive precursor was mainly incorporated into diacyl-phospholipids (phosphatidylcholine, phosphatidylethanolamine and their lyso-derivatives) as reported previously [21] (not shown) The acidic fraction corresponding to the schizont stage showed a significantly high incorporation in comparison with ring and trophozoite stages (Table 1); thus similar amounts of radioactivity of each fraction was applied to the TLC plate Acidic lipids analysed in solvent A showed main spots corresponding to phosphatidylinositol, phosphatidic acid and fatty acids (Fig 1A) The unbound fraction of each stage was treated with 0.1 M NaOH in methanol to hydrolyse non ceramide-based lipids and after purification, the samples were analysed by TLC in solvent B (Fig 1B) These lipidic components migrated close to standards of GSLs A spot with the mobility similar to a standard of sphingomyelin was also shown Even though the sample of control erythrocytes used was enhanced, only faint bands were observed In Table Incorporation of radioactive precursors (CPM per 108 parasites) in the different fractions of lipids obtained from the three intraerytrocytic stages of P falciparum C, control uninfected erythrocytes; R, rings; T, trophozoites; S, schizonts [14C]palmitic acid [14C]glucose Total lipids C R T S Neutral lipids Acidic lipids Sphingolipids Total lipids Neutral lipids Acidic lipids Sphingolipids 21800 719300 1011400 6269500 21500 457300 808300 5192900 – 5400 7400 73800 300 22800 30200 115900 3600 6047 7834 87854 2700 7300 5100 71200 – 300 400 2500 – 700 200 3800 2208 A S Couto et al (Eur J Biochem 271) Ó FEBS 2004 Fig Incorporation of [14C]palmitic acid into lipids of Plasmodium falciparum (A) TLC analysis in chloroform/methanol/water (65 : 25 : 3, v/v/v) of the acidic lipids Samples obtained from 4.8 · 107 ring forms (lane 1), 2.06 · 107 trophozoites (lane 2) and 4.38 · 106 schizonts (lane 3) were spotted in order to apply similar amounts of radioactivity PtdGr, phosphatidyl glycerol; PtdH, phosphatidic acid; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; lysoPtdIns, lysophosphatidylinositol (B) The unbound fractions of the DEAE-Sephadex column were saponified and analysed by TLC Samples corresponding to: 2.4 · 108 ring forms (lane 1); 1.0 · 108 trophozoites (lane 2); 0.4 · 108 schizonts (lane 3); 7.0 · 108 noninfected erythrocytes (lane 4) were analysed in chloroform/methanol/0.25% KCl (80 : 30 : 2, v/v/v); I, ceramide; II, glucosylceramide; III, lactosylceramide; IV, globotriaosylceramide; V, globotetraosylceramide; VI, sphingomyelin order to ensure that the labeled components corresponded to ceramide-based lipids, the spot comigrating with the standard of GbOse3Cer (IV) from the schizont fraction (Fig 1B, lane 4), was eluted from the plate and digested with ceramide glycanase As expected, hydrolysis was not complete, however, a new spot comigrating with a standard of ceramide was obtained (Fig 2A) Fig Hydrolysis of the GSLs The spot comigrating with GbOse3Cer (IV) from Fig 1B (lane 4) was incubated with ceramide glycanase for 18 h, extracted with choroform/methanol and further subjected to TLC in chloroform/metanol/0.25% KCl (80 : 30 : 2, v/v/v) Cer, ceramide; GbOse3Cer, globotriosylceramide The glycosphingolipid fraction metabolically labeled with [14C]palmitic acid was subjected to methanolysis, further treated with BF3/methanol and analysed by TLC in chloroform/metanol/1 M NH4OH (40 : 10 : 1, v/v/v).C18-Sph, C18sphingosine; C18-sSph, C18- dihydrosphingosine A sample of the saponified neutral lipids obtained from schizont stages, was further purified by Unisil column chromatography Three fractions (CHCl3, CHCl3/MeOH (98 : 2, v/v), and CHCl3/MeOH (1 : 3, v/v)) were eluted The latter, containing the glycosphingolipids, was hydrolysed with HCl/MeOH/water (3 : 29 : 4, v/v/v), treated with BF3/MeOH to methylate the rest of the fatty acids that could interfere, and analysed by TLC in solvent C (Fig 2B) Two spots, migrating in the region where long chain bases are resolved, were detected One of them (RF 0.33) with the mobility of an authentic standard of C18sphinganine, the other one (RF 0.38) migrating slightly above, would correspond to C20-sphinganine A similar result was obtained when the spot comigrating with GbOse3Cer was analysed under the same conditions (not shown) In another experiment a [14C]glucose incorporation was tried The three sugar labeled stages were extracted as above and the extracts were fractionated by DEAE-Sephadex column chromatography and saponified Although recoveries in ring and trophozoite forms extracts were low, the purified glycosphingolipid fraction obtained from the schizont stage showed a significant higher incorporation of the radiolabeled sugar than uninfected erythrocytes (Table 1) This extract was analysed by TLC in solvent B in comparison with an analogous [14C]palmitic acid labeled fraction (Fig 3A) Four spots with RF similar to those obtained by [14C]palmitic acid labeling were detected Cerebroside (RF 0.85) was clearly resolved in the sugar labeled sample (Fig 3A, lane 2) When a similar experiment was performed using [14C]galactose as precursor, a faint band corresponding to galactosylceramide was also detected (Fig 3B) In order to further analyse each GSL fraction, extracts obtained from schizont stages were fractionated as above and the neutral GSL fraction was subjected to TLC in parallel with an analogous [14C]palmitic acid labeled Ó FEBS 2004 Glycosphingolipids in Plasmodium falciparum (Eur J Biochem 271) 2209 Fig Incorporation of [14C]glucose and [14C]galactose into glycosphingolipids of Plasmodium falciparum (A) TLC analysis in chloroform/methanol/0.25% KCl (80 : 30 : 2, v/v/v) of the unbound fractions after mild alkaline treatment Lane 1, [14C]glucose labeled control erythrocytes (7 · 108 cells); lane 2, [14C]glucose labeled glycosphingolipids from schizonts (4 · 108 cells); lane 3, [14C]palmitic acid labeled glycosphingolipids from schizonts (0.4 · 108 cells) I, ceramide; II, glucosylceramide; III, lactosylceramide; IV, globotriaosylceramide; V, globotetraosylceramide; VI, sphingomyelin (B) TLC analysis in chloroform/methanol/0.25% KCl (80 : 30 : 2, v/v/v) of the glycosphingolipid fraction purified from schizonts (1.4 · 108 parasites) after metabolic incorporation of [14C]galactose GalCer, galactosylceramide fraction Spots corresponding to the ceramide fraction (I), monohexosylceramide fraction (II), trihexosylceramide fraction (IV) and tetrahexosylceramide fraction (V) were extracted from the plate and analysed by UV-MALDI-TOF MS (Fig 4) Table shows the m/z values (mass numbers) and possible sphingoid-fatty acid-sugar combinations of ceramides for the signals obtained from each fraction, taking into account the results obtained by TLC analysis of the long chain bases Presence of an active glucosylceramide synthase In a first approach, the activity of the enzyme in a parasite lysate was examined using UDP-[14C]glucose as donor and two different ceramides, palmitoyldihydrosphingosine and palmitoylsphingosine as acceptors (Fig 5A) A spot comigrating with glucosylceramide was obtained but, interestingly, although dihydroceramides are poor substrates for the mammalian enzymes [22], P falciparum enzyme seemed to be active only with the saturated compound (Fig 5A, lane 2) In order to confirm the substrate specificity of the plasmodial enzyme, the enzymatic assay was performed using fluorescent ceramides and UDP-glucose as donor Lysates from each stage were assayed and the products were analysed by TLC As expected, in the three stages, only parasite lysates incubated with BODIPY-DHCer as acceptor synthesized fluorescent glucosylceramide (Fig 5B, lanes 1–3) No fluorescent product was obtained when the unsaturated ceramide was used (Fig 5B, lanes 4–6) The special substrate specificity assures the parasite origin of the detected enzyme activity In order to show the presence of the GCS, immunoprecipitation of parasite lysates from each stage was performed using polyclonal GCS 1.2 antibody [20] The immunoprecipitates were subjected to SDS/PAGE and electrotransferred to poly(vinylidene difluoride) membranes When the membranes were developed with the GCS 5.1 antibody, a band at a molecular mass of 48 kDa was detected in the three intraerythrocytic stages (Fig 5C, lanes 2–4) Nevertheless, the possibility that the 48 kDa band can be due to a cross-reacting parasite protein not related with the GCS cannot be ruled out In mammalian cells, low concentrations of D,L-threoPPMP have no effect on sphingomyelin synthase but can inhibit the synthesis of glucosylceramides [23–26] In order to establish if the plasmodial enzyme activity was affected, the experiment was performed in the presence of lM PPMP (Fig 5D) TLC analysis revealed that the presence of threo-PPMP efficiently inhibited the synthesis of glucosylceramides (52%) Additionally, the primary effect of PPMP seemed to be specifically on GSLs, because no difference in bulk protein synthesis was seen when comparing whole [35S]methionine labeled precipitate of identical number of parasites that were left untreated or treated with lM PPMP (Fig 5D) Previous reports showed that treatment of parasite cultures with PPMP resulted in a potent inhibition of the intraerythrocytic development of P falciparum [27–30] In order to determine the effect of the inhibitor in GSLs synthesis, treatment of parasite cultures with threo-PPMP for 24 h was performed followed by incorporation of [14C]glucose or [14C]palmitic acid in the presence of the drug Parasite development was monitored by microscopy of Giemsa-stained blood smears (Table 3) As expected, treatment with threo-PPMP showed inhibition of the intraerythrocytic development at the ring stage as described by Haldar et al [26] Each labeled stage from treated and nontreated parasites was purified by Percoll gradient and further fractionated as above to achieve purified GSLs Comparison of the incorporation of [14C]glucose in the same number of treated and nontreated parasites, showed a clear reduction in the ring stage (Fig 6A) As a result of the arrest on development, a low amount of treated trophozoite and schizont stages were obtained, this fact joined to a low incorporation of the sugar precursor precluded further analysis of these stages Fractions corresponding to the same number of [14C]glucose-labeled ring forms were analysed by TLC in solvent A (Fig 6B) While the fraction obtained from nontreated ring forms showed spots corresponding to the labeled GSLs, no spots were detected in the fraction obtained from PPMP-treated ring forms As regards the palmitic acid labeled parasites, the same analysis was carried out In accordance, when the incorporation of palmitic acid was compared in treated and nontreated parasites, inhibition of the precursor incorpor- Ó FEBS 2004 2210 A S Couto et al (Eur J Biochem 271) Fig UV-MALDI-TOF mass spectra in positive ion mode of the different GSLs fractions Values indicate m/z of sodium adducted molecular ions, [M + Na]+, in nominal mass Posible ceramide species are listed in Table (A) UV-MALDI-TOF MS of ceramides (fraction I) in reflectron mode; matrix: nor-harmane; (B) UV-MALDI-TOF MS of monohexosylceramides (fraction II) in linear mode; matrix: nor-harmane; (C) UV-MALDITOF MS of globotriaosylceramides (fraction IV) in reflectron mode; matrix: 2,5-dihydroxybenzoic acid; (D) UV-MALDI-TOF MS of globotetraosylceramides (fraction V) in reflectron mode; matrix: 2,5-dihydroxybenzoic acid Table Mass numbers and possible sphingoid-fatty acid-sugar combinations of ceramides in the different fractions of GSLs obtained from schizont forms after TLC analysis Molecular related ions, [M+Na]+ are expressed as nominal mass Listed ceramide species were deduced from UV-MALDI-TOF MS spectra (Fig 4) Spectra are shown in Fig m/z, Data from UV-MALDI-TOF MS Spectra m/z Proposed structures A 494.9 522.9 537.2 550.8 553.2 569.0 d18:0-C10h:0 d20:0-C10h:0 d20:0-C10-2h:0 d18:0-C14h:0 d20:0-C10-3h:0 d18:0-C14-2h:0 522.5 550.7 568.0 656.6 686.3 d20:0-C10h:0 d18:0-C14h:0 d18:0-C14-2h:0 d18:0-C10h:0(+hex) d20:0-C10h:0 (+hex) B d18:0-C12h:0 d18:0-C12-2h:0 d20:0-C12h:0 d18:0-C12-3h:0 d20:0-C12-2h:0 d18:0-C12h:0 d20:0-C12h:0 d20:0-C12-2h:0 d18:0-C12h:0 (+hex) C 1036.5 d18:0-C14h:0(+3hex) d20:0-C12h:0(+3hex) 1052.3 d18:0-C14-2h:0(+3hex) d20:0-C12-2h:0(+3hex) 1068.3 d18:0-C14-3h:0(+3hex) d20:0-C12-3h:0(+3hex) D 1038.2 d18:0-C14h:0(+3hex) d20:0-C12h:0(+3hex) 1052.8 d18:0-C14-2h:0(+3hex) d20:0-C12-2h:0(+3hex) 1185.0 d18:0-C10h:0 (3hex+hexNAc) ation in the three stages was also observed (Fig 6C) The lipidic precursor is incorporated more efficiently probably as a result of the development of the membrane network Consequently, even when a low amount of schizont stages was obtained, the comparison on TLC could be carried out (Fig 6D) Interestingly, treated schizonts showed no glycosphingolipid components in contrast with the nontreated samples Discussion Glycosphingolipids seem to be a general feature of eukaryotic cells However, the physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in other systems [31] In this report we show for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of P falciparum Incorporation of [14C]palmitic acid and [14C]glucose allowed the analysis of purified glycosphingolipids When the long chain base component of these GSLs was investigated, using [14C]palmitic acid as a precursor, labeled sphinganine was obtained (Fig 3) in contrast with the major long chain base present in erythrocytes, indicating clearly the parasite origin of the detected compound Degradation of host sphingomyelin to produce ceramide for parasite growth has been suggested, supported by the existence of sphingomyelinase in P falciparum [30,32,33] However, although the amount of cera- Ó FEBS 2004 Glycosphingolipids in Plasmodium falciparum (Eur J Biochem 271) 2211 Fig Glucosylceramide synthase analysis (A) The enzymatic assay was performed using UDP-[14C]glucose as marker in 0.1 M sodium phosphate buffer (pH 7.4), mM b-NAD and a liposomal substrate consisting in dipalmitoylphosphatidylcholine and ceramide (10 : 1) (containing 0.1 nmol of ceramide) The mixture was purified and analysed by TLC in chloroform/methanol/water (65 : 25 : 2, v/v/v) Lane 1, palmitoylceramide; lane 2, palmitoyldihydroceramide (B) The enzyme assay was performed using the fluorescent ceramide precoupled to BSA, UDP-glucose (2.5 mM) and mM b-NAD The parasite lysates (50–100 lg protein) in 0.1 M sodium phosphate buffer (pH 7.4) The mixture was extracted with chloroform/ methanol (1 : 1) and analysed by TLC in chloroform/methanol/water (65 : 25 : 2, v/v/v) Lanes 1, and are rings, trophozoites and schizonts, respectively, using BODIPY-dihydroceramide; lanes 4, and 6, the same using BODIPY-ceramide (C) Immunoprecipitation of parasite lysates performed using polyclonal GCS 1.2 antibody The immunoprecipitates were subjected to SDS/PAGE and electrotransferred to poly(vinylidene difluoride) membranes The membranes were developed with the GCS 5.1 antibody followed by ECL 1, control erythrocytes; 2, ring forms; 3, trophozoites; 4, schizonts Molecular mass of markers is indicated (in kDa) at the right side of the figure The arrow at the left side shows the band at 48 kDa (D) The enzymatic assay was performed using schizonts as enzymatic source as in (A), without (lane 1) or with (lane 2) lM PPMP as inhibitor GlcCer, glucosylceramide; R, ring forms; T, trophozoites; S, schizonts (E) Incorporation of L-[35S]methionine in proteins obtained from parasites treated or nontreated with lM PPMP Aliquots were collected at different times (0–48 h), precipitated with 12% (w/v) trichloroacetic acid and radioactivity was measured mide produced is low, our results confirm that the de novo biosynthetic pathway of ceramides is active in this parasite Nevertheless, the last step of the ceramide biosynthesis, involving the dehydrogenation of N-acylsphinganine to N-acylsphingenine would be absent in P falciparum Additionally, [14C]galactose incorporation showed the presence of galactosylceramide as reported recently [11] The sphingolipid structure of the different products obtained in the GSL fraction was proven by UVMALDI-TOF mass spectrometry The spectra showed that the predominant components of Fraction I were ceramides involving long chain bases d18:0 or d20:0 and hydroxy fatty acids C10:0, C12:0 and C14:0 bearing one, two or three hydroxy residues (Fig 4A, Table 2) This Ó FEBS 2004 2212 A S Couto et al (Eur J Biochem 271) Table Effect of lM threo-PPMP on parasite development Parasite cultures were incubated with lM DL-threo-PPMP After 24 h of treatment, parasites were labeled with [14C]palmitic acid or [14C]glucose for 18 h in the presence of the drug Control cultures without the inhibitor were labeled under the same conditions The effect on parasite development was monitored by microscopy of Giemsa-stained blood smears in two independent experiments R, ring forms; T, trophozoites; S, schizonts [14C]glucose [14C]palmitic acid Control %R %T %S Fig Inhibition of GSL synthesis by threo-PPMP treatment GSLs were purified from threo-PPMP treated and nontreated parasites and were further analysed by TLC in chloroform/methanol/water (65 : 25 : 3, v/v/v) (A) Comparison of the radioactivity recovered in the GSL fractions obtained from treated (unfilled bars) and nontreated (filled bars) [14C]glucose incorporated parasites R, ring forms; T, trophozoites; S, schizonts; C, control uninfected erythrocytes (B) Lane 1, control [14C]glucose labeled ring stage (4.2 · 108 parasites); lane 2, threo-PPMP-treated [14C]glucose labeled ring stage (5.5 · 108 parasites) (C) Comparison of the radioactivity recovered in the GSL fractions obtained from treated (unfilled bars) and nontreated (filled bars) [14C]palmitic acid incorporated parasites R, ring forms; T, trophozoites; S, schizonts; C, control uninfected erythrocytes (D) Lane 1, control [14C]palmitic acid labeled schizonts (0.13 · 108 parasites); lane 2, threo-PPMP-treated [14C]palmitic acid labeled schizonts (0.16 · 108 parasites) In all cases, at each stage, a similar number of parasites was compared finding is in accordance with a previous report showing that the de novo biosynthetic pathway of fatty acids in P falciparum involved C10:0 to C14:0, some of them Treated Control Treated 5.0 3.1 1.4 5.2 2.7 0.1 4.5 2.7 1.5 4.4 2.1 0.1 hydroxylated [34] Fraction II migrated very near Fraction I and was shown to be a mixture of ceramides and monohexosyl ceramides, not very well resolved The latter were mainly monohexosylceramides of d18:0 and d20:0 acylated with C10h:0 and C12h:0 (Fig 4B, Table 2) Spectrum C (Fig 4) showed that the predominant component of Fraction IV is a trihexosylceramide (m/z 1036.5) with a possible sphingoid-fatty acid combination d18:0-C14h:0 (or d20:0-C12h:0) On the other hand, Fraction V (Fig 4, spectrum D) showed less intense signals than the others However, it was very interesting to detect a component of m/z 1185.0 corresponding to a tetrahexosylceramide bearing an N-acetylhexosamine residue This result agrees with the fact that incorporation of tritiated glucosamine led to the preferential detection of GSLs migrating as highly glycosylated species [10] Biosynthesis of GSLs in P falciparum pointed to the presence of an active glucosylceramide transferase When the enzyme activity was searched in parasite lysates using UDP-[14C]glucose as marker as well as using fluorescent ceramides, activity was found only when the dihydroceramide was used as substrate This is in good agreement with the result described above and would explain earlier reports showing that the parasites were not competent to the formation of glucosylceramide when using unsaturated ceramides [28] GCS from different eukaryotic kingdoms have been cloned; remarkably their sequences present only a few conserved amino acids and the overall similarity between the enzymes from species with remote evolutionary relationship is rather low [31] In particular for P falciparum, we were unable to find any sequences related to GCS This is not rare as it has been suggested that enzymes are more difficult to identify in P falciparum by sequence similarity methods The difficulty has been attributed either to the great evolutionary distance between P falciparum and other well studied organisms or to the high A + T content of the genome [35] Nevertheless, we detected a potential gene for GCS (GenBankTM, accession number NP_701286) with conserved domains for glycosyltransferases [36] However, in an attempt to detect the presence of the plasmodial enzyme in a parasite lysate, immunoprecipitation with polyclonal antibodies against the human GCS was tried A band of molecular mass near 48 kDa was recognized in the three stages of the parasite This band Ó FEBS 2004 Glycosphingolipids in Plasmodium falciparum (Eur J Biochem 271) 2213 was absent in the control erythrocytes The apparent Mr resembles the predicted molecular mass of the human and rat GCS polypeptides although the empirical molecular mass described is  38 kDa [20] In mammalian cells, low concentrations (1–5 lM) of D,Lthreo-PPMP have no effect on sphingomyelin synthase but can inhibit the synthesis of glucosylceramides In P falciparum, PPMP has been described as a potent inhibitor of the intraerythrocytic maturation leading to an arrest of the parasites at ring stage Rings formed in the presence of the drug contain no tubular structures On the contrary, mature trophozoites and schizonts that contain a fully extended tubular network were not affected by the drug [26,27,29,30] When we tried the action of PPMP in vitro on the GCS, using UDP-[14C]glucose as marker, the enzyme activity which resulted was clearly reduced (Fig 5D) In another experiment, when the inhibitor was added in parasite cultures, we observed an arrest on parasite development Parasites collected at the ring stage had been treated with PPMP at the trophozoite stage ( 40 h before), and resulted unaffected (Table 3) On the contrary, parasites collected at the schizont stage that had received the inhibitor at the ring stage, were not able to evolve and died When the [14C]glucose labeled GSL fraction purified from PPMP treated and from control parasites collected at the ring stage were compared by TLC, disappearance of GSLs was shown (Fig 6B) This fact indicates that although parasites are able to evolve to the ring stage, no new GSLs are biosynthesized Using [14C]palmitic acid as precursor, the analysis could also be performed with the schizont stage Likewise, parasites treated with PPMP showed disappearance of GSLs (Fig 6D) In this case two hypotheses may be postulated: PPMP is also acting on the glucosyltransferase and although there is de novo synthesis of ceramides, the glycosylating step is blocked; or, parasites that overcome treatment are so stressed that the tubovesicular membrane network is not able to import the lipidic precursor Anyway, the possibility of both events taking place simultaneously must be considered In conclusion, we have isolated and characterized the major GSL structures present in the intraerythrocytic forms of P falciparum by UV-MALDI-TOF mass spectrometry A glucosylceramide synthase activity with specificity for saturated ceramides which can be inhibited by low concentrations of PPMP was identified for the first time The inhibitor, used in cultures, arrests parasite development with a concomitant depletion of GSLs The special feature presented by the plasmodial GCS, joined to the expanding number of cellular functions that may be glycosphingolipid dependent, makes this enzyme a promising target for antimalarial drug development Studies are underway for the characterization of the enzyme and its intracellular location in P falciparum Acknowledgements This work was supported by grants from: CONICET, Universidad de ´ Buenos Aires and Agencia Nacional de Promocion Cientı´ fica y ´ Tecnologica (Pict 06-06545), Argentina FAPESP, CNPq, PRONEX, Brazil, UNDP/World Bank/WHO (TDR) A S C and R E.-B are members of Research Council CONICET (Argentina) and C C., ANPCyT fellow Mass spectrometry was performed as part of the Academic Agreement between R E.-B and H N with the facilities of the High Resolution Liquid Chromatography-integrated Mass Spectrometer System Laboratory of the United Graduate School of Agricultural Sciences (Ehime University, Japan) and partially supported by 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Etwiller, L., Eddy, S.R., Griffiths-Jones, S., Howe, K.L., Marshall, M & Sonnhammer, E.L (2002) The Pfam Protein Families Nucleic Acids Res 30, 276–280  ... fraction, taking into account the results obtained by TLC analysis of the long chain bases Presence of an active glucosylceramide synthase In a first approach, the activity of the enzyme in a parasite... sphingomyelin synthase but can inhibit the synthesis of glucosylceramides In P falciparum, PPMP has been described as a potent inhibitor of the intraerythrocytic maturation leading to an arrest of the... extremely important for understanding GSL function In this report, we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of P falciparum