Parasitol Res (1992) 78:416-422
Parasitology Research 9 Springer-Verlag1992
Labeling and initial characterization of polar lipids in cultures of Plasmodium falciparum * A. Dieckmann-Sehuppert 1, S. Bender 1, A.A. Holder 2, K. Haldar 3, and R.T. Schwarz 1 1 Zentrumfiir Hygieneund MedizinischeMikrobiologie,UniversitfitMarburg, Robert-Koch-Str. 17, W-3550 Marburg, Federal Republic of Germany 2 Divisionof Parasitology,National Institute for MedicalResearch, London NW7 1AA 3 Dept. of Microbiologyand Immunology,StanfordUniversity,Stanford, CA 94305, USA Accepted March 2, 1992
Abstract. The present report describes the radioactive labeling of polar lipids in in vitro cultures of Plasmodium falciparum as well as their extraction with organic solvents and their partial characterization by chemical and enzymatic methods. All substances detected could be cleaved by alkali, suggesting that they were esters rather than sphingolipids or compounds containing alkyl groups. Dolichol-cycle intermediates were not detected. Phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine were labeled by fatty acids and inositol or ethanolamine, respectively, confirming their de novo synthesis by the parasite. Metabolic labeling with glucosamine and cleavage by phosphatidylinositol-specific phospholipase C provided evidence of the formation of N-acetyl-glucosaminylphosphatidylinositol, an obligate precursor in the biosynthesis of glycosylphosphatidylinositol membrane anchors of proteins.
Lipids and glycolipids are essential membrane components as well as potential intermediates in the posttranslational modification of proteins, i.e., glycosylation, and the formation and attachment of glycosylphosphatidyl* This study was supported by grants Schw 296/4-1 and 296/4-2 from the Deutsche Forschungsgemeinschaft,by the British-German Academic Research Collaboration (ARC) Program of the German Academic Exchange Service (DAAD), by the Fonds der Chemischen Industrie, by the Hessisches Ministeriumfiir Wissenschaft und Kunst, and by the P.E. KempesFoundation,Marburg Correspondence to: A. Dieckmann-Schuppert Abbreviations: Cho, Choline; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis-(/~-aminoether)-N,N'-tetraacetic acid; EtN, ethanolamine;Fuc, fucose; Gal, galactose; Glc, glucose; GlcN, glucosamine;GlcNAc, N-acetylglucosamine;HEPES, N-2hydroxyethylpiperazinyl-N'-2-ethanesulfonicacid; HPAEC, high pH anion-exchange chromatography; Man, mannose; NP-40, Nonidet P-40; PI, phosphatidylinositol;PE, phosphatidylethanolamine; PCho, phosphatidylcholine;PMSF, phenylmethylsulfonyl fluoride; TLC, thin-layer chromatography; TLCK, tosyllysinechloromethylketone
inositol (GPI) membrane anchors. Moreover, inositolcontaining phospholipids are known to be involved in signal transduction. A significance of GPI compounds for cellular signaling has been postulated (Low and Saltiel 1988; Eardley and Koshland 1991). Plasmodium falciparum is the causative agent of human malignant malaria tropica. Despite massive efforts in vaccine and chemotherapy development, this disease presently causes the death of several million people per year (WHO 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required for the development of better chemotherapeutic and vaccination strategies. One of the neglected areas of malaria biochemistry involves the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides, be they linked to lipids or to proteins, in P. falciparurn. Glycolipids may be membrane components per se and as such may represent potential antigens or be involved in the formation of glycoproteins, e.g., dolichol-cycle or GPI-membrane anchor biosynthetic intermediates. The importance of glycolipids, which contribute to glycoprotein assembly or are themselves integrated into proteins, seems to be obvious in the light of the biochemical, topological, or immunological relevance of the glycosylation of proteins. Few proteins of P.falciparum have thus far been identified as putative glycoproteins (Howard and Reese 1984; Ramasamy 1987). The occurrence of GPI-anchored proteins in P. falciparum is suggested both by metabolic labeling (Haldar et al. 1985, 1986; Schwarz et al. 1986, 1987) and by the use of phosphatidylinositolspecific phospholipase C (Braun-Breton etal. 1988, 1990). Inhibitors of protein glycosylation can inhibit the multiplication of P. falciparum in vitro (Udeinya and van Dyke 1981 a, b; Dieckmann-Schuppert et al. 1992 a). Taken together, these observations strongly suggest the presence of side-chain-glycosylated proteins apart from the putative GPI-achored ones. We therefore embarked on a series of studies to investigate the occurrence and structure of glycolipids and glycoproteins in malaria. In the initial study reported herein, we tried to follow the
417 biosynthesis o f the c o r r e s p o n d i n g glycolipids u s i n g P.
falciparum c u l t u r e d in vitro. U s i n g m e t a b o l i c labeling, t h i n - l a y e r c h r o m a t o g r a p h i c analysis, a n d e n z y m a t i c a n d chemical t r e a t m e n t s , we showed that large a m o u n t s o f p h o s p h a t i d y l i c lipids were synthesized in culture, whereas there was a n a p p a r e n t lack of f o r m a t i o n o f dolicholcycle intermediates, c o n f i r m i n g recent findings o n protein g l y c o s y l a t i o n ( D i e c k m a n n - S c h u p p e r t et al. 1992b). The numbers and amounts of putative GPI-anchored b i o s y n t h e t i c i n t e r m e d i a t e s synthesized in c u l t u r e were small as c o m p a r e d with the results recently o b t a i n e d i n o u r l a b o r a t o r y u s i n g a cell-free system ( G e r o l d et al. 1991).
Materials and methods
In vitro culture of Plasmodium falciparum P. falciparum cloned strains T9/94 and FCR-3/A2 were maintained in RPMI 1640 medium supplemented with 25 mM HEPES, 21 mM sodium bicarbonate, 0.37 mM hypoxanthine, 0.1 mg neomycin/ml, and 10% (v/v) human A + serum (Red Cross Blood Donation Center, Frankfurt/Main). The cultures contained human A + erythrocytes (leukocyte-depleted erythrocyte concentrate; Blood Donation Center, University of Marburg) to a hematocrit of up to 5% and were incubated at 37~ C in gas-tight containers (modular incubation chamber, Flow) gassed with a mixture of 90% nitrogen and 5% each of oxygen and carbon dioxide. Development and multiplication of the cultures were followed by microscopic evaluation of Giemsa-stained thin smears. Synchronous development was achieved by repeated treatment with 5% sorbitol as described by Lambros and Vanderberg (1979). Unless specified otherwise, cultures harboring 28- to 40-h-old trophozoites were used for the present study. Metabolic labeling and lysis of the cells All radioactive substances were obtained from Amersham-Buchler (Braunschweig, FRG) and used at 100 gCi/ml. The usual labeling time was 4 h. Thereafter, the cultures were washed three times in a 10-fold excess of ice-cold phosphate-buffered saline, and the cells were then lysed in twice their volume of ice-cold lysis buffer (50 mM TRIS-HC1, pH 8.0; 5 mM each of EDTA and EGTA; 1% (w/v) Nonidet P-40; 1 mM phenylmethylsulfonyl fluoride; 5 mM iodoacetamide; 0.1 mM tosyllysine-chloromethylketone; i ~tg leupeptin/ml. The lysates were stored at - 8 0 ~ C. [3H]-Ethanolamine (29.5 Ci/mmol) was added directly to the cultures. Tritiated fatty acids (myristic, 53.5 Ci/mmol; palmitic, 55 Ci/mmol) originally supplied in ethanolic solution were dried under nitrogen, redissolved in a few microliters of 70% ethanol, and then added to normal culture medium. The final ethanol concentration in the cultures never exceeded 0.02%, which in control experiments had been shown not to affect the parasites. Tritiated sugar labeling (2-[3H]-mannose, 13.6Ci/mmol; 6-[3H]-glucos amine, 25.4Ci/mmol; 6-[3H]-galactose, 25.5 Ci/mmol; L-6-[3H]fucose, 70 Ci/mmol) was performed in glucose-free RPMI 1640 medium (Amimed, Muttenz, Switzerland) to which 10 mM fructose had been added (Schwarz et al. 1986). In this case, the cultures were preincubated in the glucose-free medium for 30 min prior to the addition of radiolabel to deplete endogenous glucose stores.
Organic solvent extraction Lipophilic substances were extracted from culture lysates with 3 times a 50-fold volume of ice-cold hexane/isopropanol (3:2, v/v;
Rosen et al. 1989). To the pooled extracts, 1/6 vol. water was added. After phase separation, the upper phase was usually devoid of radioactivity or contained the unmetabolized fatty acids, if such were used in the particular experiment. The radioactive compounds in the lower isopropanol/water phase were analyzed by TLC. Other extraction protocols (Orlandi and Turco 1987; Menon et al. 1988) were tried but proved to be less effective.
Hydrolysis procedures Monosaccharides were liberated from glycolipids by hydrolysis in 4 N hydrochloric acid at 100~ C for 4 h. Following removal of the acid by methanol evaporation and phase partition between n-butanol and water, the water-soluble compounds released were analyzed by HPAEC (see below). Inositol- or ethanolamine-labeled compounds were hydrotyzed using 6 N HC1 for 15 h at 100~ C (Wang et al. 1990) and then processed as described above. To cleave sugars from putative dotichol-cycle intermediates, the corresponding fractions were subjected to mild acid hydrolysis in 1 N HC1/ 50% n-propanol at 50~ for 15min (McDowell and Schwarz 1988). Alkaline cleavage of oxyester linkages was performed in 0.25 N KOH in methanol for 4 h at 50~ C, followed by acidification to pH 3.5 using acetic acid and subsequent butanol-water partition.
Monosaccharide and inositol analysis Monosaccharides and inositol were separated and identified by HPAEC on a Carbopac PAl column (4 x 250 mm; Bio-LC, Dionex Co., Sunnyvale, Calif.) eluted isocratically with 15 mM sodium hydroxide at a flow rate of 1.0 ml/min.
TLC analysis Glycolipids were analyzed by TLC on silica G60 plates (Merck) developed with chloroform/methanol/acetic acid/water (25:15:4: 2, by vol. ; Menon et al. 1988; system A), with chloroform/methanol/ water (65:25:4, by vol.; Lehle and Tanner 1978; system B), or with petrol ether/ethyl ether/acetic acid (70:30:1, by vol.; Kates 1986; system C). Ethanolamine and its metabolites were separated on cellulose plates (Merck) developed with n-butanol/acetic acid/ water (5: 2: 3, by vol.; Schneider 1969; system D). The radioactivity distribution was analyzed using an automatic TLC linear analyzer (Berthold, model LB2842). Single peaks were eluted with methanol for further analysis.
Phosphatidylinositol-specific phospholipase C Samples were dried under nitrogen and redissolved in 50 gl buffer (70 mM triethanolamine-HC1, 0.16 % sodium deoxycholate, pH 7.5) to which 0.5 IU enzyme (PI-PLC; EC 3.1.4.3, from Bacillus cereus, Boehringer) had been added (Mayor and Menon 1990). After 12 h incubation at 37~ C, the reaction was stopped by boiling the mixture for 2 min. A phase partition between water-saturated n-butanol and water was performed to separate hydrophilic and lipophilic reaction products. The radioactivity was determined by liquid scintillation counting of both phases, and the organic phase was analyzed by TLC. The B. cereus enzyme cleaves not only GPI structures but also phosphatidylic compounds (Menon, personal communication).
418
Phospholipase A2
Results
Samples were dried under nitrogen and redissolved in 50 Ixl buffer (100mM TRIS/HC1, 0.1% sodium deoxycholate, 1 mM CaCI2, pH 7.4) to which 50 IU enzyme (PLA2; EC 3.1.1.4, from porcine pancreas, Boehringer) had been added (Mayor and Menon 1990). After 12 h incubation at 37~ C, the reaction was stopped and further processed as described above for PI-PLC. Porcine-pancreas phospholipase Az cleaves GPI structures only poorly but acts well on other phospholipids.
The patterns o b t a i n e d b y T L C o f the various radiolabeled organic solvent extracts are s h o w n in Fig. 1 for the T9/94 strain. The patterns obtained using the F C R 3/A2 strain were identical (data n o t shown). Peaks m a r k e d with an asterisk represent the respective radiolabeled precursor c o m p o u n d s , which were used for the labeling and appeared in varying a m o u n t s in the extracts. These were identified b y c o c h r o m a t o g r a p h y with authentic standards. The highest i n c o r p o r a t i o n was obtained by fatty acid or ethanolamine labeling, whereas inositol or sugar labeling consistently yielded low recoveries in the organic extract, Total lipid extracts were subjected to acid hydrolysis and their radioactive hydrophilic constituents were analyzed by H P A E C or T L C as appropriate. The results o f these analyses are summarized in Table 1. Selected p r o m i n e n t peaks (those n u m b e r e d in Fig. 1) were eluted f r o m the T L C plate, treated with either phospholipase or nitrous acid, and r e c h r o m a t o g r a p h e d .
Deaminative cleavage by nitrous acid For the investigation of deaminative cleavage by nitrous acid (Mayor and Menon 1990), samples were dried under nitrogen and redissolved in 100 gl nitrous acid, which was freshly prepared each time by mixing equal volumes of 0.2 M acetate buffer (pH 4.0) and 0.5 M sodium nitrite. After 12 h incubation at room temperature, the reaction was stopped by the addition of 6 I11 5 N HC1 and samples were phase-partitioned and further processed as described above.
A
i
2
.~ 3 -B
F 3
C~
!
2
C~
2 9
9
C
2
Q
l .
D
~~ .
.
.
,
.
.
4 .
.
.
.
.
.
.
.
I.
4
.-.-
s
10
15
cm
_ 20
B
10
15 c m
.
~.
.
.
Fig. 1 A-H. Distribution of radioactivity on thin-layer chromatograms of organic solvent extracts from Plasmodiumfalciparum T9/ 94 cultures labeled with A tritiated inositol, B ethanolamine, C myristic acid, D palmitic acid, E galactose, F mannose, G glucosamine, and H fucose. Chromatography was performed in solvent system A and the radioactivity distribution was measured using a Berthold LB 2842 linear analyzer. Asterisks indicate the respective unmetabolized compound
419 Table 1. Identification of polar constituents of organic solvent extracts from radiolabeled Plasrnodiumfalciparum cultures Labeled precursor or isolated lipid
Identified on hydrolysis of the lipid fraction as :
Glucosamine Mannose
Glucosamine a 65% Mannose, 35% not identified a 91% Galactose, 9% not identified a 55% Fucose, 45% not identified" Inositol" Inositol a 69% Ethanolamine, 31% choline b Choline b Ethanolamine b
Galactose Fucose Inositol Putative PI (peak 1, Fig. 1 A) Ethanolamine Putative PCho (peak 2, Fig. 1 B) Putative PE (peak 4, Fig. 1 B)
Percentages represent the average of 2 determinations "Analyzed by HPAEC b Analyzed by TLC
Table 2. Sensitivity toward phospholipases and different chemical cleavage procedures of selected peaks obtained by TLC of organic solvent extracts from radiolabeled Plasmodiumfalciparum cultures Labeled precursor
Peak Sensitivity toward number a PLA2 PIPLC HNO2 Mild Alkali acid
Palmitic acid
1 2 3 4 5 Myristic acid 1 2 3 Ethanolamine 1 2 3 Inositol Galactose
Mannose
Glucosamine
Fuco se
+ + + + + + + + + + +
+ + + + + + + + + + +
+ -
-
4
+
+
-
-
+
1
+
+
-
-
+
1 2 3 1 2 3 1 2 3 4 a
(+) . . (+) . . (+) (+) (+) (+) (+ )
+
-
+
-
+ + + +
-
+ + + + + + + + + + +
+ . .
. .
. .
. .
. .
+ . . + + + + +
+ + + + + + + + + + +
a Refers to the numbering in Fig. 1 Parentheses denote partial cleavage
The results o f these treatments are summarized in Table 2. Mild acid hydrolysis designed to cleave dolicholp y r o p h o s p h a t e saccharides did n o t affect the position or shape o f a n y o f these peaks.
Besides free inositol, the only c o m p o u n d detected in the inositol-labeled extract (peak 1 in Fig. 1 A) was identified as phosphatidylinositol (PI) by c o c h r o m a t o g r a p h y with diacyl-PI in solvent systems A and B as well as by liberation o f free inositol, which was identified by H P A E C u p o n acid hydrolysis o f the isolated c o m p o u n d (cf. Table I). A c o m p o u n d exhibiting the same T L C m o bility in solvent systems A and B, thus presumably also representing parasite-synthesized PI, was also labeled by myristic and palmitic acids (peak 2 in Figs. 1 C, D). U n infected erythrocytes did n o t synthesize inositol-labeled PI (data n o t shown). The total inositol-labeled extract contained only inositol as shown by H P A E C analysis. E t h a n o l a m i n e was i n c o r p o r a t e d by the parasite culture into at least eight c o m p o u n d s (Fig. 1 B), two o f which (peaks 2 and 4) could be identified as phosphatidylcholine (PCho) and p h o s p h a t i d y l e t h a n o l a m i n e (PE), respectively, by c o c h r o m a t o g r a p h y with d i a c y l - P C h o or diacyl-PE in solvent systems A and B. O n acid hydrolysis, p e a k 2 released choline and p e a k 4 e t h a n o l a m i n e (cf. Table 1), which were identified by T L C in solvent system D. Like PI, PE and P C h o were also labeled by myristic and palmitic acids (peaks 1 and 3, and 1 and 4 in Figs. 1 C a n d 1 D, respectively). The total ethanolamine-labeled extract also contained only choline and e t h a n o l a m i n e as evidenced by T L C in system D but showed no evidence o f other metabolites o f ethanolamine. Choline, choline phosphate, a n d C D P - c h o l i n e were also detected in perchloric acid supernatants f r o m ethanolamine-labeled Plasmodium falciparum cultures ( D i e c k m a n n - S c h u p p e r t et al. 1992 b). Myristic acid was n o t i n c o r p o r a t e d (Fig. 1 C) into a n y other detectable c o m p o u n d besides the putative PI, P C h o , and PE. The radioactivity at the f r o n t likely represents free fatty acid as j u d g e d f r o m its mobility in T L C system D. A p a r t f r o m its a b o v e - m e n t i o n e d presence in the putative P C h o , PE, a n d PI, palmitic acid was i n c o r p o r a t e d into two other peaks (3 and 5 in Fig. 1 D), which were n o t f o u n d to be labeled with myristate. D u e to their mobility o n T L C and to their susceptibility to P L A 2 and P I P L C treatment being identical with those o f peaks 1, 2, and 4, these two peaks could eventually represent differentially acylated m i n o r species o f either one o f the previously m e n t i o n e d phosphatidylic c o m p o u n d s . Peak 6 p r o b a b l y represents free fatty acid for the reasons stated above. The highest i n c o r p o r a t i o n rates a m o n g the radioactive sugars were obtained using galactose. It labeled three m a j o r peaks (Fig. 1 E), the third o f which showed a mobility similar to that o f the mannose-labeled peak 2 (Fig. 1 F). B o t h glucosamine a n d fucose were incorporated into a n u m b e r o f c o m p a r a b l y small peaks (Figs. 1 G and 1 H, respectively). Only the sugar-labeled peaks remaining at the p o i n t o f application to the plate (peak 1 in Fig. 1 E - H ) and the glucosamine-labeled peaks 2 and 4 exhibited sensitivity not only t o w a r d the phospholipases used but also t o w a r d nitrous acid (cf. Table 2) and m i g h t therefore possess G P I structures. Moreover, m o s t radioactivity except that o f the aforem e n t i o n e d peaks 1, the small glucosamine-labeled p e a k 3, and the fraction migrating at the front, partitioned
420 into the aqueous phase on phase-partitioning between water-saturated n-butanol and water of the previously dried extract. Since butanol-water partitioning can be quite efficiently used to purify GPI precursors (Menon et al. 1988), this again suggests that such compounds had indeed been formed, albeit in small amounts. Discussion
This report shows that a variety of lipophilic compounds can be radioactively labeled in in vitro cultures of Plasmodiumfalciparum and describes the extraction and partial characterization of some of these substances. The formation of these compounds must have been accomplished by the parasite, as the presence of leukocytes, which could also have been responsible for the incorporation, was excluded on several occasions by microscopic inspection of the blood used for culture after selective lysis of the red cells with 3% acetic acid. Moreover, leukocytes were never seen on Giemsa-stained slides. Inositol was incorporated only into PI, and exclusively so by parasitized cultures. Uninfected erythrocytes do not synthesize PI because they do not possess the corresponding enzyme. The low amount of PI present in the normal red cell membrane is presumably acquired from the serum. Moreover, the PI content of erythroeytes rises significantly on infection with P. falciparum (Vial and Ancelin 1992). Interestingly, no inositol was found incorporated into compounds other than PI. In parallel with the above-mentioned increase observed in PI content on infection, an even greater increase is seen in PE and PCho (Vial and Ancelin 1992). This is reflected in the incorporation by the parasite of radioactive ethanolamine into PE and PCho, indicating a de novo synthesis of these compounds. A de novo biosynthesis of PE from ethanolamine by the parasite has been described by Vial et al. (1984) and by Ancelin and Vial (1986). The formation by the parasite of PCho via methylation of PE (the Bremer pathway) has been found in the same laboratory (Vial et al. 1982). Labeling by glucosamine of a number of uncharacterized malarial glycolipids and their extraction with a mixture of chloroform and methanol has previously been reported by other investigators (Sherwood et al. 1986, 1988), albeit for another strain (Malayan Camp strain from infected Aotus monkeys). The TLC pattern shown by these authors is not comparable with ours, presumably due not only to the use of different strains but rather to different labeling, extraction, and separation conditions. Sherwood et al. (1986) reported the recovery of unmetabolized glucosamine (GlcN) after acid hydrolysis of their glucosamine-labeled glycolipids. This finding was confirmed in the present study. In contrast to higher eukaryotic cells (Warren 1972) and to the more closely related apicomplexan parasite Toxoplasma gondii (Odenthal:Schnittler et al., unpublished data), which readily metabolize glucosamine to galactosamine and other sugars;: malaria parasites show no detectable conversion of glucosamine apart from its N-acetylation. Therefore, P. falciparum may not possess an active uri-
dine diphosphate (UDP)-N-acetyl-D-glucosamine-4'-epimerase system. This hypothesis is further supported by our recovery of exclusively unmetabolized glucosamine or N-acetylglucosamine not only from organic solvent extracts but also from proteins and from perchloric acid supernatants obtained from glucosamine-labeled P. falciparum cultures (Dieckmann-Schuppert et al. 1992b). The PI-PLC and partial PLA2 sensitivity of the glucosamine-labeled peak 3 (Fig. 1 G, Table 2) and its resistance to nitrous acid cleavage suggest that this compound may be malarial N-acetylglucosamine-PI. The ability of P. falciparum to synthesize glucosamine-PI as well was recently shown in our laboratory using a cellfree system prepared from isolated plasmodia (Gerold et al. 1991). In contrast to Sherwood et al. (1986), we did succeed in demonstrating the incorporation of a small amount of radioactivity from fucose into the lipid extract, of which about 45% was recovered as a compound eluting at the same position as galactosamine on HPAEC; however, taking into account the probable metabolic pathways involved, this substance is not likely to be galactosamine. Besides fucose, galactose and mannose were also partially recovered in metabolized form from the organic solvent extracts. In all, 9% of the galactose label was converted to a compound that again showed the same elution properties as galactosamine on HPAEC, but for the same reasons as stated above, this compound is unlikely to represent the latter. The unidentified 2-[3H] mannose metabolite is not mannosamine as evidenced by its comparison with standards. The sensitivity to alkali of all compounds described herein suggests that these substances are not sphingolipids nor do they carry an alkyl group. Alkyl linkages have been shown to occur in a glycolipid, in the lipophosphoglycan, and in the anchor structure of a GPIanchored protein of Leishmania parasites (Field et al. 1991; Schneider et al. 1990; Orlandi and Turco 1987). On alkali treatment, the compounds described herein released all radioactivity either as free fatty acid (in the case of fatty acid labeling) or into the aqueous phase on butanol-water partitioning (in the other cases). All of these substances should therefore be assumed to exhibit binding of their lipid moiety via an oxyester bond. The presence of thioesters can be excluded because of the insensitivity of the compounds to hydroxylamine treatment (Magee 1988). The occurrence of sphingolipids in other lower eukaryotes had thus far been conclusively shown only for yeast and other fungi (reviewed by Stults et al. 1989), and one report each suggests the presence of sphingolipids in the flagellates Trypanosoma mega (order: Trypanosomatidae; Vermelho et al. 1986) and Tritrichomonas foetus (order: Trichomonadida; Singh et al. 1991). The membrane of the malaria parasite's host erythrocyte is very rich in different glycosphingolipids (Hanfland 1975; Stults et al. 1989). On infection of the erythrocyte with P. falciparum, the total sphingolipid content seems either to remain unchanged (Vial and Ancelin 1992) or to decrease (Maguire and Sherman 1990). Since there is evidence for the existence of sphingomyelin synthetase in P. falciparum (Haldar et al.
421 1991), some synthesis of sphingomyelin should occur if the total levels remain the same. This observation does not conflict with our failure to detect any acid-labile metabolite of ethanolamine (i.e., sphingomyelin), since the labeling efficacy or the amount of radiolabel present may not have sufficed for detection. Following mild acid hydrolysis, no dolichol-cycle intermediates were detected among the peaks shown in Fig. 1. This observation is in accordance with other results suggesting an apparent lack of N-glycosylation in asexual intraerythrocytic malaria parasites (DieckmannSchuppert et al. 1992b). Mbaya et al. (1990) investigated isoprenoid metabolism in intraerythrocytic P. falciparum and found that farnesyl-pyrophosphate was synthesized from labeled mevalonate but did not obtain evidence of the formation o f dolichol itself. However, this would not exclude the formation of small amounts of dolichol from these compounds or the use of dolichols from the erythrocyte lipids. P. falciparum was shown in our laboratory to be capable of synthesizing dolichol- or isoprenoid-phosphate-mannose and -glucose from radioactive exogenous GDP-mannose or UDP-glucose, respectively (Gerold et al. 1991), but the formation of Dol-PP-Nacetylglucosamine has not been observed to date. The incorporation of radioactive sugars into lipophilic compounds synthesized by P. falciparum during our 4-h labeling period and extracted into the organic solvent was rather low. Longer labeling times were avoided so as to circumvent detrimental effects on the parasites of glucose starvation, which might have occurred despite the substitution with fructose. Moreover, longer labeling in the presence of the normal glucose concentration would raise the possibility of the radioactive sugars undergoing more extensive metabolic rearrangement and/ or glycolytic degradation such that the labeling would no longer be specific. In addition, if intracellular pools are large, labeling with the exogenously added substances should be expected to be poor. Poor labeling of GPI structures is a well-known phenomenon in mammalian cells. Moreover, the several membrane barriers surrounding the intraerythrocytic P. falciparum could lead to trapping of polar radioactive precursors in a compartment outside the parasite. Regardless of the relatively low amount of radioactivity incorporated in the substances described in the present report, we did obtain positive evidence of the ability of P. falciparum to synthesize N-acetylglucosamine-PI, which is a prerequisite for the synthesis of larger GPI structures. Since at least some putative GPI-anchored proteins have been described for P. faleiparum (Haldar et al. 1985, 1986; Schwarz et al. 1987; Braun-Breton etal. 1988, 1990), biosynthetic intermediates or precursors such as those characterized in trypanosomes (Menon et al. 1988; Krakow et al. 1989) should be formed, although they may be poorly labeled for any of the reasons given above. Bearing this in mind and considering the extremely low incorporation of even palmitic acid into compounds other than PI, PE, or PCho as well as our failure to detect significant amounts of PI-PLC- and nitrous acid-sensitive compounds apart from the putative GlcNAc-PI and the sugar-labeled peaks 1 (Fig. 1 E-H),
we therefore established a cell-free system prepared from isolated P. falciparum and began studying the synthesis of malarial glycolipids using this system rather than cultures. This approach has led to the initial identification and characterization of some larger GPI structures (Gerold et al. 1991). Glycolipids may carry antigenic epitopes. Such immunogenic glycolipids are known to occur in other parasitic protozoa such as Leishmania (Turco 1988) and T. gondii (Striepen et al. 1991). Frankenburg et al. (1984) have found glycolipids from the rodent malaria parasite P. berghei to be antigenic. Antigenic glycolipids of P. falciparum have been described by Taverne et al. (1990) and Sj6berg et al. (1991). In our experiments, we have thus far not succeeded in demonstrating the presence of antigenic components in P. falciparum cultures by immunostaining on T L C plates using different sera from malaria-positive subjects. This work is being continued with the use of additional antisera. The compounds detected in the present study may be structural membrane components, precursors in glycolipid synthesis, or lipid intermediates in other pathways. In any case, the biosynthesis of these lipids may use enzymes unique to malarial parasites, and such knowledge may therefore lead to the identification of new antimalarial agents.
Acknowledgement. Prof. V. Kretschmer (University Blood Bank, Marburg) kindly provided human erythrocytes for the in vitro cultivation of Plasmodiumfalciparum.
References
Ancelin ML, Vial HJ (1986) Several lines of evidence demonstrating that Plasmodiumfalciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine. FEBS Lett 202: 217-223 Braun-Breton C, Rosenberry TL, Pereira da Silva L (1988) Induction of the proteolytic activity of a membrane protein in Plasmodiumfalciparum by phosphatidyl inositol-specific phospholipase C. Nature 332:457-459 Braun-Breton C, Rosenberry TL, Pereira da Silva LH (1990) Glycolipid anchorage of Plasmodiumfalciparum surface antigens. Res Immunol 141:743-755 Dieckmann-Schuppert A, Hensel J, Schwarz RT (1992a) Studies on the effect of Tunicamycin on Plasmodiumfalciparum. Biochem Soc Trans 20:184S Dieckmann-Schuppert A, Bender S, Odenthal-Schnittler M, Bause E, Schwarz RT (1992b) Apparent lack of N-Glycosylation in the asexual intraerythrocytic stage of Plasmodiumfalciparum. Eur J Biochem (in press) Eardley DD, Koshland ME (1991) Glycosylphosphatidylinositol: a candidate system for interleukin-2 signal transduction. Science 251:78-81 Field MC, Medina-Acosta E, Cross GAM (1991) Characterization of a glycosylphosphatidylinositol membrane protein anchor precursor in Leishmania mexicana. Mol Biomed Parasitol 48 : 227-230 Frankenburg S, Slutzky GM, Gitler C, Londner MV (1984) Lipid antigens derived from erythrocytes infected with Plasmodium berghei. Z Parasitenkd 70:331-336 Gerold P, Dieckmann-Schuppert A, Schwarz RT (1991) Glycolipids synthesized by a cell-free system prepared from the malaria parasite, Plasmodium falciparum. Biol Chem Hoppe Seyler 372:661 662
422 Haldar K, Ferguson MAJ, Cross GAM (1985) Acylation of a Plasmodium falciparum merozoite surface antigen via sn-l,2-diacylglycerol. J Biol Chem 260:4969-4974 Haldar K, Henderson CL, Cross GAM (1986) Identification of the parasite transferrin receptor of Plasmodium falciparum infected erythrocytes and its acylation via 1,2-diacyl-sn-glycerol. Proc Natl Acad Sci USA 83:8565-8569 Haldar K, Uyetake L, Ghori N, Elmendorf HG, Li WL (1991) The accumulation and metabolism of a fluorescent ceramide derivative in Plasmodiumfaleiparum-infected erythrocytes. Mol Biochem Parasitol 49: 143-156 Hanfland P (1975) Characterization of B and H blood-group active glycosphingolipids from human B erythrocyte membranes. Chem Phys Lipids 15 : 105-124 Howard RF, Reese RT (1984) Synthesis of merozoite proteins and glycoproteins synthesized during the schizogony of Plasmodium falciparum. Mol Biochem Parasitol i0: 319-334 Kates M (1986) Techniques of lipidology, 2nd edn. Elsevier, Amsterdam Krakow JL, Doering TL, Masterson WJ, Hart GW, Englund PT (1989) A glycolipid from Trypanosoma brucei related to the variant surface glycoprotein membrane anchor. Mol Biochem Parasitol 36: 263-270 Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium faleiparum erythrocytic stages in culture. J Parasito165:418-420 Lehle L, Tanner W (1978) Glycosyl transfer from dolichol-phosphate sugars to endogenous and exogenous glycoprotein acceptors in yeast. Eur J Biochem 83 : 563-570 Low MG, Saltiel AR (1988) Structural and functional roles of glycosyl-phosphatidylinositolin biological membranes. Science 239: 268-275 Magee AI (1988) Identification and characterization of fatty acidacylated proteins in cultured cells by radiolabeling. In: Brodbeck U, Bordier C (eds) Post-translational modifications of proteins by lipids. Springer, Berlin Heidelberg New York, pp 59-63 Maguire PA, Sherman IW (1990) Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells. Mol Biochem Parasitol 38:105-112 Mayor S, Menon AK (1990) Structural analysis of the glycoinositol phospholipid anchors of membrane proteins. Methods: a Companion to Methods Enzymol 1 : 297-305 Mbaya B, Rigomier D, Edorh G, Karst F, Schrevel J (1990) Isoprenoid metabolism in Plasmodium faleiparum during the intraerythroeytic phase of malaria. Biochem Biophys Res Commun 173:849-854 McDowell W, Schwarz RT (1988) Lipid-mediated protein glycosylation: assembly of lipid-linked oligosaccharides and posttranslational oligosaccharide trimming. In: Brodbeck U, Bordier C (eds) Post-translational modifications of proteins by lipids. Springer, Berlin Heidelberg New York, pp 99-118 Menon AK, Mayor S, Ferguson MAJ, Duszenko M, Cross GAM (1988) Candidate glycolipid precursor for the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brueei variant surface glycoproteins. J Biol Chem 263:1970-1977 Orlandi PA, Turco SJ (1987) Structure of the lipid moiety of the Leishmania donovani lipophosphoglycan. J Biol Chem 262:10384-10391 Ramasamy R (1987) Studies on glycoproteins in the human malaria parasite Plasmodiumfalciparum. Immunol Cell Biol 65:147-152 Rosen G, Pahlsson P, Londner MV, Westerman ME, Nilson AL (1989) Structural analysis of glycosyl-phosphatidylinositolantigens of Leishmania major. J Biol Chem 264:9043-9052 Schneider P, Ferguson MAJ, McConville MJ, Mehlert A, Homans SW, Bordier C (1990) Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease. J Biol Chem 265:16955-16964 Schneider WC (1969) Enzymatic preparation of labeled phosphorylcholine, phosphorylethanolamine, cytidine diphosphate choline, deoxycytidine diphosphate choline, cytidine diphosphate
ethanolamine, and deoxycytidine diphosphate ethanolamine. Methods Enzymol 14:684-690 Schwarz RT, Riveros-Moreno V, Lockyer M J, Nicholls SC, Davey LS, Hillman Y, Sandhu JS, Freeman RR, Holder AA (1986) Structural diversity of the major surface antigen of Plasmodium falciparum merozoites. Mol Cell Biol 6:964-968 Schwarz RT, Lockyer MJ, Holder AA (1987) Aspects of the posttranslational modification of a major Plasmodium falciparum merozoite surface antigen. In: Chang K-P, Snary D (eds) Hostparasite cellular and molecular interactions. NATO ASI Ser HI 1 : 275-279 Sherwood JA, Spitalnik SL, Aley SB, Quakyi IA, Howard RJ (1986) Plasmodiumfalciparum and P. knowlesi: initial identification and characterization of malaria synthesized glycolipids. Exp Parasitol 62:127-141 Sherwood JA, Spitalnik SL, Suarez SC, Marsh K, Howard RJ (1988) Plasmodium faleiparum glycolipid synthesis: constant and variant molecules of isolates and of strains with differing knob and cytoadherence phenotype. J Protozool 35:169-172 Singh BN, Costello CE, Beach DH (1991) Structures of glyeophosphosphingolipids of Tritrichomonasfoetus: a novel glycophosphophingolipid. Arch Biochem Biophys 286:409-418 Sj6berg K, Hosein Z, Wfihlin B, Carlsson J, Wahlgren M, TroyeBlomberg M, Berzins K, Perlman P (199t) Plasmodiumfalciparurn: an invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen. Exp Parasitol 73 : 317-325 Striepen B, Tomavo S, Dubremetz JF, Schwarz RT (1991) The "low molecular weight antigen" of Toxoplasma gondii is a family of glycosylphosphatidylinositol lipids. Biol Chem Hoppe Seyler 372: 765 Stults CLM, Sweeley CC, Macher BA (1989) Glycosphingolipids: structure, biological source, and properties. Methods Enzymol 179:167-214 Taverne J, Bate CAW, Playfair JHL (1990) Malaria exoantigens induce TNF, are toxic and are blocked by T-independent antibody. Immunol Lett 25: 207-212 Turco SJ (1988) The lipophosphoglycan of Leishmania. Parasitol Today 4:255-257 Udeinya IJ, Dyke K van (1981a) 2-Deoxyglucose: inhibition of parasitemia and of glucosamine incorporation into glycosylated macromolecules, in malarial parasites (Plasmodiumfalciparum). Pharmacology 23:165-170 Udeinya I J, Dyke K van (1981 b) Concurrent inhibition by Tunicamycin of glycosylation and parasitemia in malarial parasites (Plasmodiumfaleiparum) cultured in human erythrocytes. Pharmacology 23 : 171-175 Vermelho AB, Hogge L, Barreto-Bergter E (1986) Isolation and characterization of a neutral glycosphingolipid from the epimastigote form of Trypanosoma mega. J,Protozool 33:208-231 Vial H, Ancelin ML (1992) Malarial lipids: an overview. Subcell Biochem (in press) Vial HJ, Thuet MJ, Philippot JR (1982) Phospholipid biosynthesis in synchronous Plasmodium faleiparum cultures. J Protozool 29:258-263 Vial HJ, Thuet MJ, Philippot JR (1984) Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers. Biochim Biophys Acta 795 : 372-383 Wang WT, Safar J, Zopf D (1990) Analysis of inositol by highperformance liquid chromatography. Anal Biochem 188:432435 Warren L (1972) The biosynthesis and metabolism of amino sugars and amino sugar-containing heterosaccharides. In: Gottschalk A (ed) Glycoproteins, 2nd edn. Elsevier, Amsterdam, pp 10971126 WHO (1989) Ninth programme report of the UNDP/World Bank/ WHO-TDR. World Health Organization, Geneva
Parasitology Research 9 Springer-Verlag1992
Labeling and initial characterization of polar lipids in cultures of Plasmodium falciparum * A. Dieckmann-Sehuppert 1, S. Bender 1, A.A. Holder 2, K. Haldar 3, and R.T. Schwarz 1 1 Zentrumfiir Hygieneund MedizinischeMikrobiologie,UniversitfitMarburg, Robert-Koch-Str. 17, W-3550 Marburg, Federal Republic of Germany 2 Divisionof Parasitology,National Institute for MedicalResearch, London NW7 1AA 3 Dept. of Microbiologyand Immunology,StanfordUniversity,Stanford, CA 94305, USA Accepted March 2, 1992
Abstract. The present report describes the radioactive labeling of polar lipids in in vitro cultures of Plasmodium falciparum as well as their extraction with organic solvents and their partial characterization by chemical and enzymatic methods. All substances detected could be cleaved by alkali, suggesting that they were esters rather than sphingolipids or compounds containing alkyl groups. Dolichol-cycle intermediates were not detected. Phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine were labeled by fatty acids and inositol or ethanolamine, respectively, confirming their de novo synthesis by the parasite. Metabolic labeling with glucosamine and cleavage by phosphatidylinositol-specific phospholipase C provided evidence of the formation of N-acetyl-glucosaminylphosphatidylinositol, an obligate precursor in the biosynthesis of glycosylphosphatidylinositol membrane anchors of proteins.
Lipids and glycolipids are essential membrane components as well as potential intermediates in the posttranslational modification of proteins, i.e., glycosylation, and the formation and attachment of glycosylphosphatidyl* This study was supported by grants Schw 296/4-1 and 296/4-2 from the Deutsche Forschungsgemeinschaft,by the British-German Academic Research Collaboration (ARC) Program of the German Academic Exchange Service (DAAD), by the Fonds der Chemischen Industrie, by the Hessisches Ministeriumfiir Wissenschaft und Kunst, and by the P.E. KempesFoundation,Marburg Correspondence to: A. Dieckmann-Schuppert Abbreviations: Cho, Choline; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis-(/~-aminoether)-N,N'-tetraacetic acid; EtN, ethanolamine;Fuc, fucose; Gal, galactose; Glc, glucose; GlcN, glucosamine;GlcNAc, N-acetylglucosamine;HEPES, N-2hydroxyethylpiperazinyl-N'-2-ethanesulfonicacid; HPAEC, high pH anion-exchange chromatography; Man, mannose; NP-40, Nonidet P-40; PI, phosphatidylinositol;PE, phosphatidylethanolamine; PCho, phosphatidylcholine;PMSF, phenylmethylsulfonyl fluoride; TLC, thin-layer chromatography; TLCK, tosyllysinechloromethylketone
inositol (GPI) membrane anchors. Moreover, inositolcontaining phospholipids are known to be involved in signal transduction. A significance of GPI compounds for cellular signaling has been postulated (Low and Saltiel 1988; Eardley and Koshland 1991). Plasmodium falciparum is the causative agent of human malignant malaria tropica. Despite massive efforts in vaccine and chemotherapy development, this disease presently causes the death of several million people per year (WHO 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required for the development of better chemotherapeutic and vaccination strategies. One of the neglected areas of malaria biochemistry involves the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides, be they linked to lipids or to proteins, in P. falciparurn. Glycolipids may be membrane components per se and as such may represent potential antigens or be involved in the formation of glycoproteins, e.g., dolichol-cycle or GPI-membrane anchor biosynthetic intermediates. The importance of glycolipids, which contribute to glycoprotein assembly or are themselves integrated into proteins, seems to be obvious in the light of the biochemical, topological, or immunological relevance of the glycosylation of proteins. Few proteins of P.falciparum have thus far been identified as putative glycoproteins (Howard and Reese 1984; Ramasamy 1987). The occurrence of GPI-anchored proteins in P. falciparum is suggested both by metabolic labeling (Haldar et al. 1985, 1986; Schwarz et al. 1986, 1987) and by the use of phosphatidylinositolspecific phospholipase C (Braun-Breton etal. 1988, 1990). Inhibitors of protein glycosylation can inhibit the multiplication of P. falciparum in vitro (Udeinya and van Dyke 1981 a, b; Dieckmann-Schuppert et al. 1992 a). Taken together, these observations strongly suggest the presence of side-chain-glycosylated proteins apart from the putative GPI-achored ones. We therefore embarked on a series of studies to investigate the occurrence and structure of glycolipids and glycoproteins in malaria. In the initial study reported herein, we tried to follow the
417 biosynthesis o f the c o r r e s p o n d i n g glycolipids u s i n g P.
falciparum c u l t u r e d in vitro. U s i n g m e t a b o l i c labeling, t h i n - l a y e r c h r o m a t o g r a p h i c analysis, a n d e n z y m a t i c a n d chemical t r e a t m e n t s , we showed that large a m o u n t s o f p h o s p h a t i d y l i c lipids were synthesized in culture, whereas there was a n a p p a r e n t lack of f o r m a t i o n o f dolicholcycle intermediates, c o n f i r m i n g recent findings o n protein g l y c o s y l a t i o n ( D i e c k m a n n - S c h u p p e r t et al. 1992b). The numbers and amounts of putative GPI-anchored b i o s y n t h e t i c i n t e r m e d i a t e s synthesized in c u l t u r e were small as c o m p a r e d with the results recently o b t a i n e d i n o u r l a b o r a t o r y u s i n g a cell-free system ( G e r o l d et al. 1991).
Materials and methods
In vitro culture of Plasmodium falciparum P. falciparum cloned strains T9/94 and FCR-3/A2 were maintained in RPMI 1640 medium supplemented with 25 mM HEPES, 21 mM sodium bicarbonate, 0.37 mM hypoxanthine, 0.1 mg neomycin/ml, and 10% (v/v) human A + serum (Red Cross Blood Donation Center, Frankfurt/Main). The cultures contained human A + erythrocytes (leukocyte-depleted erythrocyte concentrate; Blood Donation Center, University of Marburg) to a hematocrit of up to 5% and were incubated at 37~ C in gas-tight containers (modular incubation chamber, Flow) gassed with a mixture of 90% nitrogen and 5% each of oxygen and carbon dioxide. Development and multiplication of the cultures were followed by microscopic evaluation of Giemsa-stained thin smears. Synchronous development was achieved by repeated treatment with 5% sorbitol as described by Lambros and Vanderberg (1979). Unless specified otherwise, cultures harboring 28- to 40-h-old trophozoites were used for the present study. Metabolic labeling and lysis of the cells All radioactive substances were obtained from Amersham-Buchler (Braunschweig, FRG) and used at 100 gCi/ml. The usual labeling time was 4 h. Thereafter, the cultures were washed three times in a 10-fold excess of ice-cold phosphate-buffered saline, and the cells were then lysed in twice their volume of ice-cold lysis buffer (50 mM TRIS-HC1, pH 8.0; 5 mM each of EDTA and EGTA; 1% (w/v) Nonidet P-40; 1 mM phenylmethylsulfonyl fluoride; 5 mM iodoacetamide; 0.1 mM tosyllysine-chloromethylketone; i ~tg leupeptin/ml. The lysates were stored at - 8 0 ~ C. [3H]-Ethanolamine (29.5 Ci/mmol) was added directly to the cultures. Tritiated fatty acids (myristic, 53.5 Ci/mmol; palmitic, 55 Ci/mmol) originally supplied in ethanolic solution were dried under nitrogen, redissolved in a few microliters of 70% ethanol, and then added to normal culture medium. The final ethanol concentration in the cultures never exceeded 0.02%, which in control experiments had been shown not to affect the parasites. Tritiated sugar labeling (2-[3H]-mannose, 13.6Ci/mmol; 6-[3H]-glucos amine, 25.4Ci/mmol; 6-[3H]-galactose, 25.5 Ci/mmol; L-6-[3H]fucose, 70 Ci/mmol) was performed in glucose-free RPMI 1640 medium (Amimed, Muttenz, Switzerland) to which 10 mM fructose had been added (Schwarz et al. 1986). In this case, the cultures were preincubated in the glucose-free medium for 30 min prior to the addition of radiolabel to deplete endogenous glucose stores.
Organic solvent extraction Lipophilic substances were extracted from culture lysates with 3 times a 50-fold volume of ice-cold hexane/isopropanol (3:2, v/v;
Rosen et al. 1989). To the pooled extracts, 1/6 vol. water was added. After phase separation, the upper phase was usually devoid of radioactivity or contained the unmetabolized fatty acids, if such were used in the particular experiment. The radioactive compounds in the lower isopropanol/water phase were analyzed by TLC. Other extraction protocols (Orlandi and Turco 1987; Menon et al. 1988) were tried but proved to be less effective.
Hydrolysis procedures Monosaccharides were liberated from glycolipids by hydrolysis in 4 N hydrochloric acid at 100~ C for 4 h. Following removal of the acid by methanol evaporation and phase partition between n-butanol and water, the water-soluble compounds released were analyzed by HPAEC (see below). Inositol- or ethanolamine-labeled compounds were hydrotyzed using 6 N HC1 for 15 h at 100~ C (Wang et al. 1990) and then processed as described above. To cleave sugars from putative dotichol-cycle intermediates, the corresponding fractions were subjected to mild acid hydrolysis in 1 N HC1/ 50% n-propanol at 50~ for 15min (McDowell and Schwarz 1988). Alkaline cleavage of oxyester linkages was performed in 0.25 N KOH in methanol for 4 h at 50~ C, followed by acidification to pH 3.5 using acetic acid and subsequent butanol-water partition.
Monosaccharide and inositol analysis Monosaccharides and inositol were separated and identified by HPAEC on a Carbopac PAl column (4 x 250 mm; Bio-LC, Dionex Co., Sunnyvale, Calif.) eluted isocratically with 15 mM sodium hydroxide at a flow rate of 1.0 ml/min.
TLC analysis Glycolipids were analyzed by TLC on silica G60 plates (Merck) developed with chloroform/methanol/acetic acid/water (25:15:4: 2, by vol. ; Menon et al. 1988; system A), with chloroform/methanol/ water (65:25:4, by vol.; Lehle and Tanner 1978; system B), or with petrol ether/ethyl ether/acetic acid (70:30:1, by vol.; Kates 1986; system C). Ethanolamine and its metabolites were separated on cellulose plates (Merck) developed with n-butanol/acetic acid/ water (5: 2: 3, by vol.; Schneider 1969; system D). The radioactivity distribution was analyzed using an automatic TLC linear analyzer (Berthold, model LB2842). Single peaks were eluted with methanol for further analysis.
Phosphatidylinositol-specific phospholipase C Samples were dried under nitrogen and redissolved in 50 gl buffer (70 mM triethanolamine-HC1, 0.16 % sodium deoxycholate, pH 7.5) to which 0.5 IU enzyme (PI-PLC; EC 3.1.4.3, from Bacillus cereus, Boehringer) had been added (Mayor and Menon 1990). After 12 h incubation at 37~ C, the reaction was stopped by boiling the mixture for 2 min. A phase partition between water-saturated n-butanol and water was performed to separate hydrophilic and lipophilic reaction products. The radioactivity was determined by liquid scintillation counting of both phases, and the organic phase was analyzed by TLC. The B. cereus enzyme cleaves not only GPI structures but also phosphatidylic compounds (Menon, personal communication).
418
Phospholipase A2
Results
Samples were dried under nitrogen and redissolved in 50 Ixl buffer (100mM TRIS/HC1, 0.1% sodium deoxycholate, 1 mM CaCI2, pH 7.4) to which 50 IU enzyme (PLA2; EC 3.1.1.4, from porcine pancreas, Boehringer) had been added (Mayor and Menon 1990). After 12 h incubation at 37~ C, the reaction was stopped and further processed as described above for PI-PLC. Porcine-pancreas phospholipase Az cleaves GPI structures only poorly but acts well on other phospholipids.
The patterns o b t a i n e d b y T L C o f the various radiolabeled organic solvent extracts are s h o w n in Fig. 1 for the T9/94 strain. The patterns obtained using the F C R 3/A2 strain were identical (data n o t shown). Peaks m a r k e d with an asterisk represent the respective radiolabeled precursor c o m p o u n d s , which were used for the labeling and appeared in varying a m o u n t s in the extracts. These were identified b y c o c h r o m a t o g r a p h y with authentic standards. The highest i n c o r p o r a t i o n was obtained by fatty acid or ethanolamine labeling, whereas inositol or sugar labeling consistently yielded low recoveries in the organic extract, Total lipid extracts were subjected to acid hydrolysis and their radioactive hydrophilic constituents were analyzed by H P A E C or T L C as appropriate. The results o f these analyses are summarized in Table 1. Selected p r o m i n e n t peaks (those n u m b e r e d in Fig. 1) were eluted f r o m the T L C plate, treated with either phospholipase or nitrous acid, and r e c h r o m a t o g r a p h e d .
Deaminative cleavage by nitrous acid For the investigation of deaminative cleavage by nitrous acid (Mayor and Menon 1990), samples were dried under nitrogen and redissolved in 100 gl nitrous acid, which was freshly prepared each time by mixing equal volumes of 0.2 M acetate buffer (pH 4.0) and 0.5 M sodium nitrite. After 12 h incubation at room temperature, the reaction was stopped by the addition of 6 I11 5 N HC1 and samples were phase-partitioned and further processed as described above.
A
i
2
.~ 3 -B
F 3
C~
!
2
C~
2 9
9
C
2
Q
l .
D
~~ .
.
.
,
.
.
4 .
.
.
.
.
.
.
.
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4
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s
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15
cm
_ 20
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.
~.
.
.
Fig. 1 A-H. Distribution of radioactivity on thin-layer chromatograms of organic solvent extracts from Plasmodiumfalciparum T9/ 94 cultures labeled with A tritiated inositol, B ethanolamine, C myristic acid, D palmitic acid, E galactose, F mannose, G glucosamine, and H fucose. Chromatography was performed in solvent system A and the radioactivity distribution was measured using a Berthold LB 2842 linear analyzer. Asterisks indicate the respective unmetabolized compound
419 Table 1. Identification of polar constituents of organic solvent extracts from radiolabeled Plasrnodiumfalciparum cultures Labeled precursor or isolated lipid
Identified on hydrolysis of the lipid fraction as :
Glucosamine Mannose
Glucosamine a 65% Mannose, 35% not identified a 91% Galactose, 9% not identified a 55% Fucose, 45% not identified" Inositol" Inositol a 69% Ethanolamine, 31% choline b Choline b Ethanolamine b
Galactose Fucose Inositol Putative PI (peak 1, Fig. 1 A) Ethanolamine Putative PCho (peak 2, Fig. 1 B) Putative PE (peak 4, Fig. 1 B)
Percentages represent the average of 2 determinations "Analyzed by HPAEC b Analyzed by TLC
Table 2. Sensitivity toward phospholipases and different chemical cleavage procedures of selected peaks obtained by TLC of organic solvent extracts from radiolabeled Plasmodiumfalciparum cultures Labeled precursor
Peak Sensitivity toward number a PLA2 PIPLC HNO2 Mild Alkali acid
Palmitic acid
1 2 3 4 5 Myristic acid 1 2 3 Ethanolamine 1 2 3 Inositol Galactose
Mannose
Glucosamine
Fuco se
+ + + + + + + + + + +
+ + + + + + + + + + +
+ -
-
4
+
+
-
-
+
1
+
+
-
-
+
1 2 3 1 2 3 1 2 3 4 a
(+) . . (+) . . (+) (+) (+) (+) (+ )
+
-
+
-
+ + + +
-
+ + + + + + + + + + +
+ . .
. .
. .
. .
. .
+ . . + + + + +
+ + + + + + + + + + +
a Refers to the numbering in Fig. 1 Parentheses denote partial cleavage
The results o f these treatments are summarized in Table 2. Mild acid hydrolysis designed to cleave dolicholp y r o p h o s p h a t e saccharides did n o t affect the position or shape o f a n y o f these peaks.
Besides free inositol, the only c o m p o u n d detected in the inositol-labeled extract (peak 1 in Fig. 1 A) was identified as phosphatidylinositol (PI) by c o c h r o m a t o g r a p h y with diacyl-PI in solvent systems A and B as well as by liberation o f free inositol, which was identified by H P A E C u p o n acid hydrolysis o f the isolated c o m p o u n d (cf. Table I). A c o m p o u n d exhibiting the same T L C m o bility in solvent systems A and B, thus presumably also representing parasite-synthesized PI, was also labeled by myristic and palmitic acids (peak 2 in Figs. 1 C, D). U n infected erythrocytes did n o t synthesize inositol-labeled PI (data n o t shown). The total inositol-labeled extract contained only inositol as shown by H P A E C analysis. E t h a n o l a m i n e was i n c o r p o r a t e d by the parasite culture into at least eight c o m p o u n d s (Fig. 1 B), two o f which (peaks 2 and 4) could be identified as phosphatidylcholine (PCho) and p h o s p h a t i d y l e t h a n o l a m i n e (PE), respectively, by c o c h r o m a t o g r a p h y with d i a c y l - P C h o or diacyl-PE in solvent systems A and B. O n acid hydrolysis, p e a k 2 released choline and p e a k 4 e t h a n o l a m i n e (cf. Table 1), which were identified by T L C in solvent system D. Like PI, PE and P C h o were also labeled by myristic and palmitic acids (peaks 1 and 3, and 1 and 4 in Figs. 1 C a n d 1 D, respectively). The total ethanolamine-labeled extract also contained only choline and e t h a n o l a m i n e as evidenced by T L C in system D but showed no evidence o f other metabolites o f ethanolamine. Choline, choline phosphate, a n d C D P - c h o l i n e were also detected in perchloric acid supernatants f r o m ethanolamine-labeled Plasmodium falciparum cultures ( D i e c k m a n n - S c h u p p e r t et al. 1992 b). Myristic acid was n o t i n c o r p o r a t e d (Fig. 1 C) into a n y other detectable c o m p o u n d besides the putative PI, P C h o , and PE. The radioactivity at the f r o n t likely represents free fatty acid as j u d g e d f r o m its mobility in T L C system D. A p a r t f r o m its a b o v e - m e n t i o n e d presence in the putative P C h o , PE, a n d PI, palmitic acid was i n c o r p o r a t e d into two other peaks (3 and 5 in Fig. 1 D), which were n o t f o u n d to be labeled with myristate. D u e to their mobility o n T L C and to their susceptibility to P L A 2 and P I P L C treatment being identical with those o f peaks 1, 2, and 4, these two peaks could eventually represent differentially acylated m i n o r species o f either one o f the previously m e n t i o n e d phosphatidylic c o m p o u n d s . Peak 6 p r o b a b l y represents free fatty acid for the reasons stated above. The highest i n c o r p o r a t i o n rates a m o n g the radioactive sugars were obtained using galactose. It labeled three m a j o r peaks (Fig. 1 E), the third o f which showed a mobility similar to that o f the mannose-labeled peak 2 (Fig. 1 F). B o t h glucosamine a n d fucose were incorporated into a n u m b e r o f c o m p a r a b l y small peaks (Figs. 1 G and 1 H, respectively). Only the sugar-labeled peaks remaining at the p o i n t o f application to the plate (peak 1 in Fig. 1 E - H ) and the glucosamine-labeled peaks 2 and 4 exhibited sensitivity not only t o w a r d the phospholipases used but also t o w a r d nitrous acid (cf. Table 2) and m i g h t therefore possess G P I structures. Moreover, m o s t radioactivity except that o f the aforem e n t i o n e d peaks 1, the small glucosamine-labeled p e a k 3, and the fraction migrating at the front, partitioned
420 into the aqueous phase on phase-partitioning between water-saturated n-butanol and water of the previously dried extract. Since butanol-water partitioning can be quite efficiently used to purify GPI precursors (Menon et al. 1988), this again suggests that such compounds had indeed been formed, albeit in small amounts. Discussion
This report shows that a variety of lipophilic compounds can be radioactively labeled in in vitro cultures of Plasmodiumfalciparum and describes the extraction and partial characterization of some of these substances. The formation of these compounds must have been accomplished by the parasite, as the presence of leukocytes, which could also have been responsible for the incorporation, was excluded on several occasions by microscopic inspection of the blood used for culture after selective lysis of the red cells with 3% acetic acid. Moreover, leukocytes were never seen on Giemsa-stained slides. Inositol was incorporated only into PI, and exclusively so by parasitized cultures. Uninfected erythrocytes do not synthesize PI because they do not possess the corresponding enzyme. The low amount of PI present in the normal red cell membrane is presumably acquired from the serum. Moreover, the PI content of erythroeytes rises significantly on infection with P. falciparum (Vial and Ancelin 1992). Interestingly, no inositol was found incorporated into compounds other than PI. In parallel with the above-mentioned increase observed in PI content on infection, an even greater increase is seen in PE and PCho (Vial and Ancelin 1992). This is reflected in the incorporation by the parasite of radioactive ethanolamine into PE and PCho, indicating a de novo synthesis of these compounds. A de novo biosynthesis of PE from ethanolamine by the parasite has been described by Vial et al. (1984) and by Ancelin and Vial (1986). The formation by the parasite of PCho via methylation of PE (the Bremer pathway) has been found in the same laboratory (Vial et al. 1982). Labeling by glucosamine of a number of uncharacterized malarial glycolipids and their extraction with a mixture of chloroform and methanol has previously been reported by other investigators (Sherwood et al. 1986, 1988), albeit for another strain (Malayan Camp strain from infected Aotus monkeys). The TLC pattern shown by these authors is not comparable with ours, presumably due not only to the use of different strains but rather to different labeling, extraction, and separation conditions. Sherwood et al. (1986) reported the recovery of unmetabolized glucosamine (GlcN) after acid hydrolysis of their glucosamine-labeled glycolipids. This finding was confirmed in the present study. In contrast to higher eukaryotic cells (Warren 1972) and to the more closely related apicomplexan parasite Toxoplasma gondii (Odenthal:Schnittler et al., unpublished data), which readily metabolize glucosamine to galactosamine and other sugars;: malaria parasites show no detectable conversion of glucosamine apart from its N-acetylation. Therefore, P. falciparum may not possess an active uri-
dine diphosphate (UDP)-N-acetyl-D-glucosamine-4'-epimerase system. This hypothesis is further supported by our recovery of exclusively unmetabolized glucosamine or N-acetylglucosamine not only from organic solvent extracts but also from proteins and from perchloric acid supernatants obtained from glucosamine-labeled P. falciparum cultures (Dieckmann-Schuppert et al. 1992b). The PI-PLC and partial PLA2 sensitivity of the glucosamine-labeled peak 3 (Fig. 1 G, Table 2) and its resistance to nitrous acid cleavage suggest that this compound may be malarial N-acetylglucosamine-PI. The ability of P. falciparum to synthesize glucosamine-PI as well was recently shown in our laboratory using a cellfree system prepared from isolated plasmodia (Gerold et al. 1991). In contrast to Sherwood et al. (1986), we did succeed in demonstrating the incorporation of a small amount of radioactivity from fucose into the lipid extract, of which about 45% was recovered as a compound eluting at the same position as galactosamine on HPAEC; however, taking into account the probable metabolic pathways involved, this substance is not likely to be galactosamine. Besides fucose, galactose and mannose were also partially recovered in metabolized form from the organic solvent extracts. In all, 9% of the galactose label was converted to a compound that again showed the same elution properties as galactosamine on HPAEC, but for the same reasons as stated above, this compound is unlikely to represent the latter. The unidentified 2-[3H] mannose metabolite is not mannosamine as evidenced by its comparison with standards. The sensitivity to alkali of all compounds described herein suggests that these substances are not sphingolipids nor do they carry an alkyl group. Alkyl linkages have been shown to occur in a glycolipid, in the lipophosphoglycan, and in the anchor structure of a GPIanchored protein of Leishmania parasites (Field et al. 1991; Schneider et al. 1990; Orlandi and Turco 1987). On alkali treatment, the compounds described herein released all radioactivity either as free fatty acid (in the case of fatty acid labeling) or into the aqueous phase on butanol-water partitioning (in the other cases). All of these substances should therefore be assumed to exhibit binding of their lipid moiety via an oxyester bond. The presence of thioesters can be excluded because of the insensitivity of the compounds to hydroxylamine treatment (Magee 1988). The occurrence of sphingolipids in other lower eukaryotes had thus far been conclusively shown only for yeast and other fungi (reviewed by Stults et al. 1989), and one report each suggests the presence of sphingolipids in the flagellates Trypanosoma mega (order: Trypanosomatidae; Vermelho et al. 1986) and Tritrichomonas foetus (order: Trichomonadida; Singh et al. 1991). The membrane of the malaria parasite's host erythrocyte is very rich in different glycosphingolipids (Hanfland 1975; Stults et al. 1989). On infection of the erythrocyte with P. falciparum, the total sphingolipid content seems either to remain unchanged (Vial and Ancelin 1992) or to decrease (Maguire and Sherman 1990). Since there is evidence for the existence of sphingomyelin synthetase in P. falciparum (Haldar et al.
421 1991), some synthesis of sphingomyelin should occur if the total levels remain the same. This observation does not conflict with our failure to detect any acid-labile metabolite of ethanolamine (i.e., sphingomyelin), since the labeling efficacy or the amount of radiolabel present may not have sufficed for detection. Following mild acid hydrolysis, no dolichol-cycle intermediates were detected among the peaks shown in Fig. 1. This observation is in accordance with other results suggesting an apparent lack of N-glycosylation in asexual intraerythrocytic malaria parasites (DieckmannSchuppert et al. 1992b). Mbaya et al. (1990) investigated isoprenoid metabolism in intraerythrocytic P. falciparum and found that farnesyl-pyrophosphate was synthesized from labeled mevalonate but did not obtain evidence of the formation o f dolichol itself. However, this would not exclude the formation of small amounts of dolichol from these compounds or the use of dolichols from the erythrocyte lipids. P. falciparum was shown in our laboratory to be capable of synthesizing dolichol- or isoprenoid-phosphate-mannose and -glucose from radioactive exogenous GDP-mannose or UDP-glucose, respectively (Gerold et al. 1991), but the formation of Dol-PP-Nacetylglucosamine has not been observed to date. The incorporation of radioactive sugars into lipophilic compounds synthesized by P. falciparum during our 4-h labeling period and extracted into the organic solvent was rather low. Longer labeling times were avoided so as to circumvent detrimental effects on the parasites of glucose starvation, which might have occurred despite the substitution with fructose. Moreover, longer labeling in the presence of the normal glucose concentration would raise the possibility of the radioactive sugars undergoing more extensive metabolic rearrangement and/ or glycolytic degradation such that the labeling would no longer be specific. In addition, if intracellular pools are large, labeling with the exogenously added substances should be expected to be poor. Poor labeling of GPI structures is a well-known phenomenon in mammalian cells. Moreover, the several membrane barriers surrounding the intraerythrocytic P. falciparum could lead to trapping of polar radioactive precursors in a compartment outside the parasite. Regardless of the relatively low amount of radioactivity incorporated in the substances described in the present report, we did obtain positive evidence of the ability of P. falciparum to synthesize N-acetylglucosamine-PI, which is a prerequisite for the synthesis of larger GPI structures. Since at least some putative GPI-anchored proteins have been described for P. faleiparum (Haldar et al. 1985, 1986; Schwarz et al. 1987; Braun-Breton etal. 1988, 1990), biosynthetic intermediates or precursors such as those characterized in trypanosomes (Menon et al. 1988; Krakow et al. 1989) should be formed, although they may be poorly labeled for any of the reasons given above. Bearing this in mind and considering the extremely low incorporation of even palmitic acid into compounds other than PI, PE, or PCho as well as our failure to detect significant amounts of PI-PLC- and nitrous acid-sensitive compounds apart from the putative GlcNAc-PI and the sugar-labeled peaks 1 (Fig. 1 E-H),
we therefore established a cell-free system prepared from isolated P. falciparum and began studying the synthesis of malarial glycolipids using this system rather than cultures. This approach has led to the initial identification and characterization of some larger GPI structures (Gerold et al. 1991). Glycolipids may carry antigenic epitopes. Such immunogenic glycolipids are known to occur in other parasitic protozoa such as Leishmania (Turco 1988) and T. gondii (Striepen et al. 1991). Frankenburg et al. (1984) have found glycolipids from the rodent malaria parasite P. berghei to be antigenic. Antigenic glycolipids of P. falciparum have been described by Taverne et al. (1990) and Sj6berg et al. (1991). In our experiments, we have thus far not succeeded in demonstrating the presence of antigenic components in P. falciparum cultures by immunostaining on T L C plates using different sera from malaria-positive subjects. This work is being continued with the use of additional antisera. The compounds detected in the present study may be structural membrane components, precursors in glycolipid synthesis, or lipid intermediates in other pathways. In any case, the biosynthesis of these lipids may use enzymes unique to malarial parasites, and such knowledge may therefore lead to the identification of new antimalarial agents.
Acknowledgement. Prof. V. Kretschmer (University Blood Bank, Marburg) kindly provided human erythrocytes for the in vitro cultivation of Plasmodiumfalciparum.
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