EXPERIMENTALPARASITOLOGY
74,251-260(1992)
donovani: Purification and Partial Characterization Glycophosphosphingolipid Antigen Expressed on Promastigote Surface
Leishmania
of a
TRIPTI DE-MAJUMDAR Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta - 700 032, India DE-MAJUMDAR, T. 1992.Leishmania donovani: Purification and partial characterization of a glycophosphosphingolipid antigen expressed on promastigote surface. Experimental Parasitology 74,251-260. A ceramide-anchored glycophosphosphingolipid antigen was isolated from the lipid extract of Leishmania donovani promastigotes. The aflinity-purified glycolipid antigen contained galactose, mannose, myo-inositol, phosphate, ceramide, and hexosamine but no sialic acid. The phosphate group was present internally at the core of the structure: inositol (l-O)phosphorylceramide. The phosphate group became susceptible to alkaline phosphatase only after alkali-catalyzed hydrolysis of the glycolipid. o 1s~ Academic press, IIIC. INDEX DESCRIPTORS AND ABBREVIATIONS: Leishmania donovani; Kala-azar; glycophosphosphingolipid; membrane antigen; ceramide anchor; GIPL, glycophosphatidylinositol lipids; GSPL, glycophosphosphingolipid of Leishmania donovani; RIA, radioimmunoassay; RCA-I, Ricinus communis agglutinin; PMSF, phenylmethylsulfonyl fluoride; H:DE, hexane:diethyl ether; C:M, chloroform:methanol; GlcNH,, glucosamine; GLC, gas-liquid chromatography; TFA, trifluoroacetic acid; PBS, phosphate-buffered saline; Solvent A, chloroform:methanol:ethyl acetate:pyridine:4.5 N ammonia:water (15:15:5:0.5:0.5:0.5, v/v); Solvent B, acetone:diethyl ether:acetic acid (30:70:2, v/v); Solvent C, chloroform: methanol:0.25 N ammonium hydroxide in 0.25% KC1 (65:45:9, v/v); Solvent D, 0.01 M phosphate buffer, pH 6.4, containing 0.05 N ammonium hydroxide and 0.1% sodium salt of taurodeoxycholic acid; Solvent E, pyridine:ethyl acetate:acetic acid:0.25% KC1 (36:36:7:21, v/v); Solvent F, I-butanol:pyridine:0.25% KC1 (3:2:1, v/v).
of antigenic determinants (Yamakawa and Nagai 1978; Cullis and De-Kruijff 1979). A Leishmania donovani is a protozoan par- class of GIPLs, anchored to the cell memasite that causes visceral leishmaniasis or brane via diacyl glycerols, alkyl acyl glycKala-azar, a disease widely prevalent in erols, lysoalkyl glycerols, or ceramides, many parts of the tropical world and gener- has been found in several eukaryotic cells ally fatal if allowed to go untreated (WHO (Ferguson and Williams 1988), including 1984). Little is known about how the para- the parasitic protozoan Leishmania (Handsite is recognized by the host macrophages man et al. 1984; Handman and Goding and how it avoids destruction, although in 1985; King et al. 1987; McConville et al. recent years it is becoming increasingly ev- 1987, 1990; Orlandi and Salvatoro 1987; ident that surface glycoconjugates, includ- Turco et al. 1987; Rosen et al. 1989; Jaffe et ing glycolipids of pathogens, play a crucial al. 1990). The glycophosphosphingolipids conrole in host-parasite interactions or in protection against them (Kato 1973; Berman taining a phosphodiester linkage between and Dwyer 1981; Handman et al. 1986, inositol and ceramide (inositol (l-O)1987; Turco 1988; McConville et al. 1987; phosphoryl-(O-l) ceramide) are a class of GIPLs in which the lipid is anchored to the Rosen et al. 1989). cell membrane via ceramides. These have Lipids, which are essential structural components of biological membranes, play been described mainly in plants, yeasts, and fungi, and it has been suggested that a crucial role in the cell surface recognition, the cell-cell interaction, and the expression these may be functional analogues of the INTRODUCTION
251 OOl4-4894/92$5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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gangliosides found in the animal cell plasma membrane (Laine and Hich 1987). Recently, similar compounds have been reported in the parasitic protozoans Trypanosoma cruzi (Previato et al. 1990) and Tritrichonomonasfoetus (Beach et al. 1990) and also in the slime mold Dictyostelium discoideum (Stadler et al. 1989), but to date, no GSPLs have been reported from any of the Leishmania species studied so far. A ceramide-anchored inositol sphingolipid has been reported from L. donovani, but it is not glycosylated (Kaneshiro et al. 1986). This paper reports the purification and partial characterization of a ceramideanchored GIPL antigen from the L. donovani promastigote surface membrane. The potential biological role of the antigen is discussed. MATERIALS
AND METHODS
Leishmania parasites. Leishmania strain UR6 (MHOIIN/1978/UR6) was isolated from an Indian patient with Kala-azar (Datta et al. 1987). The organisms were maintained and grown in modified Ray’s medium (Ray 1932) and subcultures were made at 72-hr intervals. For radiolabeling studies, parasites were grown in the semidefined medium of Chaudhury et al. (1986). Preparation of antisera. Antibodies against the whole-cell promastigote homogenates were prepared by four subcutaneous injections into rabbits (2 x IO6 promastigotes). Samples of the extracted glycolipid were tested for antigenicity by a RIA, according to the method of Basu et al. (1987) and 2-18 ng of antigen was taken in each well. Lacto-neo-pentaglycosylceramide was used as a negative control (Basu and Basu 1973). Metabolic labeling ofparasites. Late log phase promastigotes (2 x 108) from blood agar cultures were washed three times with PBS and were biosynthetitally labeled with 500 pCi [32P]orthophosphate in 5 ml of a phosphate-free medium of Chaudhury et al. (1986), with 100 pCi of [‘Hlmannose or [3H]galactose in 5 ml of a glucose-free medium (Chaudhury et al. 1986), with 10 nCi of myo-[U-‘4C]inositol, with 10 nCi of [l-‘4C]palmitic acid, or with 100 nCi of D-[6-3H]GlcNH,, for 3 hr at room temperature. External labeling of the cell surface carbohydrates was done according to the method of Ghambarg and Hakomori (1973). Radiolabeled parasites were collected, washed in PBS, and extracted with Solvent A, and the glycolipid was purified as described in the text.
Preparation of a promastigote membrane devoid of internal material and jlagella. Ghosts from L. donovani promastigotes were prepared according to a
method developed in our laboratory (Sarkar and Bhaduri 1987). Briefly, late log phase (1 g wet wt) flagellated promastigotes were taken up in 50 mM Tris-HCl, pH 7.4, containing 2 mM PMSF and were kept on ice for 2 hr with occasional vortexing. Cells were collected by centrifugation and the pellet was washed extensively with PBS. The partially purified membrane fraction from a discontinuous sucrose density gradient centrifugation [5%(2 m1)/25%(2 ml), w/v] was further purified on a second density gradient. This purified preparation was used throughout. Extraction and purification of glycophosphosphingolipid. Late log phase promastigotes (1 g wet wt)
were washed with PBS and extracted with 19 vol of Solvent A. The extracted material was concentrated by rotary evaporation and loaded onto a column of DEAE-Sephadex A-25 (0.5 x 5 cm) equilibrated in Solvent D. The column was washed with 5 ml of the solvent, and the anionic glycolipids were eluted with a gradient of KC1 in Solvent D. The fraction containing the GSPL antigen was precipitated with 1 vol of acetone. After 3 days at 4°C to allow the precipitate to settle, the supernatant was siphoned off. The precipitate was washed with acetone and dried. The dried precipitate was taken up in 1 ml of C:M (9: 1, v/v) and loaded onto a silicic acid column preequilibrated in chloroform (10 g silicic acid/g lipid). The column was sequentially eluted with 10 bed vol each of chloroform and then with C:M (8:2; 7:3; 6:4; 1:l; 4:6; 3:7; 2:8; v/v) and finally with methanol. The fraction containing the GSPL antigen (C:M; 4:6; v/v) was further purified on a RCA-I-Sepharose 4B affinity column (0.8 x 8 cm, equilibrated in Solvent D). The column was washed with 10 bed vol of Solvent D. and the adsorbed material was eluted with 5 ml of 0.1 M galactose in Solvent D. The galactose eluent was dialyzed against PBS at 4°C for 8 hr with frequent changes of buffer and finally lyophilized. Each fraction was monitored for the presence of the glycolipid antigen by TLC in Solvent C and autoradiography. Acid hydrolysis. After acid hydrolysis of GSPL, neutral sugar was estimated by the phenol-sulfuric acid method of Dubois et al. (1956). Hexosamine was estimated according to the method of Reissing et a/. (1955) and sialic acid according to the method of Svennerholm (1958). Phosphorus was assayed by the Bartlett (1959) method, and long chain bases were quantitated according to the method of Lauter and Trams (1962) after acidic methanolysis of GSPL. Sphingosine was used as standard. Anion-exchange chromatography. The purified GSPL was subjected to anion-exchange chromatography on a DEAE-Sephadex A-25 column (0.5 x 5 cm), and the bound material eluted with a linear gradient of
Leishmania donovani ANTIGEN KC1 (O-O.1it4) in Solvent D. Fractions of 500 ~1 were collected and counted for radioactivity in a liquid scintillation counter. The GSPL was also digested with alkaline phosphatase, before and after treatment with mild alkali (1 M KOH, 18 hr, 37°C) and subjected to anion-exchange chromatography as described for the untreated GSPL. Mild acid hydrolysis under controlled conditions followed by anion-exchange chromatography. Mild acid
hydrolysis of the myo-[i4C]inositol-labeled GSPL was carried out under controlled conditions. GSPL (18320 cpm/lOO pl) was treated with 2 N HCl(100 ~1)for 1 hr at 100°C. After removal of HCl, the hydrolyzate was subjected to anion-exchange chromatography on a DEAE-Sephadex A-25 column (0.5 x 5 cm), and the material eluted with a linear gradient of KC1 (O-O.1 W in Solvent D. The eluted material was further subjected to anion-exchange chromatography under identical conditions after treatment with alkaline phosphatase. Mild alkali treatment. Mild alkali treatment of GSPL was carried out with 1 M KOH, for 18 hr at 37°C (Smith and Lester 1974). After neutralization with acetic acid, the nonpolar material was extracted with hexane. The aqueous layer was desalted on a Bio-Gel P-2 column (1.0 x 50 cm), and the material eluted in the void volume was subjected to TLC in Solvent C and exposed to autoradiography. The eluted material was also analyzed before and after treatment with alkaline phosphatase for the presence of phosphorus, galactose, mannose, GlcNH,, and inositol. Saponification ofGSPL. GSPL (1 mg) was refluxed for 8 hr in 10 ml of a mixture of acetic acid:acetic anhydride (3:2). After saponification in 6 N KOH in 95% alcohol to a final concentration of 2 N KOH, the glycosides and other nonsaponifiable products were removed by ether extraction. The saponified mixture was loaded onto a silicic acid column and after the fatty acid was eluted with H:DE (3:1), the alkyl glycerols were removed by H:DE (1:3). Both the eluents were subjected to TLC on silica gel G in Solvent B and visualized by H,SO, charring. Phospholipase C treatment. Purified GSPL (9.96 X lo5 cpm of [3H]galactose, 10.44 x lo5 cpm of [3H]mannose, or 10.34 X lo4 cpm of [14C]palmitate) was incubated with phospholipase C from Bacillus cereus (0.05 p&ml) in 50 pl of 25 mM Hepes, pH 7.5, for 1 hr at 37°C. Polar materials were extracted with water, subjected to TLC in Solvent C, and exposed to autoradiography. The digestion of the cell surface GSPL by the enzyme was monitored by using [3H]galactose-labeled parasite lysate. Parasite lysates (5 x 10scells, 1 ml, 25 mM Hepes, pH 7.5) were subjected to enzymatic digestion (0.05 &ml of enzyme) for varying periods of time and after the cells were washed free of the enzyme, the GSPL was purified as described in the text. Enzyme boiled for 5 min was used as a negative control.
253
GLC analysis of neutral sugar. After hydrolysis of samples with 2 M TFA for 4 hr at lOO”C, the neutral sugars were converted to their alditol acetates (Albersheim et al. 1%7) and analyzed by GLC on a 6-ft column of 3% ECNSS-M with a temperature program of 2Wmin from 160 to 250°C at a flow rate of 20 ml/min of carrier gas. GLC analysis of inositol and amino sugars. Inositol and GlcNH, present in the GSPL were analyzed by GLC after hydrolysis with 3 M HCl in methanol for 18 hr at 80°C. The methanolysates were dried under a stream of nitrogen, and the resulting material was dissolved in 1.0 ml of aqueous 6 M HCl and heated at 105°C for 18 hr. Fatty acids were extracted with hexame and the aqueous layer was lyophilized. The lyophilized residue was reduced with sodium borohydride and acetylated with acetic anhydride-pyridine (Albersheim et al. 1967), and the products were analyzed by GLC as described for neutral sugar analysis. GLC analysis of fatty acids. GSPL (500 kg) was methanolyzed with 0.5 M HCI in methanol (1 ml) for 18 hr at 80°C. After hexane extraction, the hexane layer was analyzed for the fatty acid methyl esters by GLC on a 6ft column of 3% ECNSS-M with a temperature program of 2”Clmin from 135to 202°C at a flow rate of 25 ml/min carrier N, gas.
RESULTS
Approximately 1 g (wet wt) of L. donovani promastigotes was extracted exhaustively with Solvent A (Fig. 1). The extracted glycolipids were fractionated on an anion exchanger, and the anionic lipids thus obtained were further separated on a silicic acid column according to their polarity. Three phosphoinositol lipid-containing fractions were obtained. The C:M (4:6) fraction showed the presence of GSPL antigen along with a closely moving band. The other two fractions showed the presence of glyceryl ethers. The GSPL antigen finally bound to the RCA-I-Sepharose affinity column and was selectively eluted with 0.1 M galactose in Solvent D. During the purification process, each fraction was monitored by TLC and visualized by exposing the TLC plates to I, vapor. Purified GSPL moved as a single band in three different solvent systems (Solvents C, E, and F) (Fig. 2). This material could be metabolically labeled with GlcNH,, galactose, man-
254
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DE-MAJUMDAR
Late log phase promastigotes 1 Promastigote ghosts Extraction with 19 vol of I Solvent A DEAE-Sephadex A-25 column (0.5 x 5 cm) O-O.1 M KC1 gradient I GSPL containing fraction precipitated with acetone I Precipitate dissolved in C:M (9: 1; v/v) 1 Silicic acid column C:M (4:6; v/v)
I Lectin-Sepharose affinity column (0.8 x 9 cm) 0.1 M galactose in I Solvent D(5 ml) Purified GSPL FIG. 1. Scheme of extraction and purification of the glycolipid.
nose, myo-inositol, palmitic acid, and phosphate. The GSPL could also be surface labeled by the galactose oxidase/NaB3H, procedure (Table I) (Ghamberg and Hakomori 1973). To establish the linkage of the phosphoinositol group to the ceramide, GSPL was subjected to mild aqueous alkaline hydrolysis under controlled conditions (1 M
4 2 3 1 FIG. 2. TLC of the radiolabeled purified GSPL. Lane 1, [3H]galactose; Lane 2, [3H]mannose; Lane 3, ‘*P-labeled affinity-purified GSPL antigen; Lane 4, 32P-labeled C:M (4:6) fraction from silicic acid column (20,000 cpm of 3H- and 500 cpm of “P-labeled compound). Chromatograms were developed in Solvent C and subjected to autoradiography.
TABLE I Metabolic and Surface Labeling Studies of L. donovani promastigotes
Metabolite Mannose Galactose Inositol Palmitic acid Phosphate Galactose oxidase/NaB3H,
Percentage of radioactivity” incorporated into purified GSPL 2.0 2.3 2.1 4.6 0.35 0.5
’ Mean percentage incorporation of radioactivity of three individual determinations.
KOH for 18 hr at 37”C), conditions that would hydrolyze inositol phosphoceramide, with the formation of a cyclic inosito1 phosphate intermediate, finally producing an inositol monophosphate and ceramide (Smith and Lester 1974). On methanolysis, the hexane layer showed the presence of fatty acid and sphingosine. The aqueous layer moved as a single band on TLC in Solvent C (Fig. 3A) and showed the presence of galactose, mannose, inositol, GlcNH,, and phosphate. These results suggest that mild alkali treatment cleaves the ceramide moiety of GSPL, with the release of the corresponding phosphoinositol oligosaccharide in the aqueous layer.
FIG. 3. TLC of the phosphoinositol oligosaccharide obtained from GSPL. (A) [3H]galactose-labeled oligosaccharide obtained by mild alkaline hydrolysis of GSPL, (B) [3H]galactose-labeled oligosaccharide obtained by phospholipase C treatment of GSPL. Chromatograms were developed in Solvent C and subjected to autoradiography.
Leishmania
donovani
The effect of phospholipase C (B. cereus) on both the purified and the membranebound GSPL was examined. The polar material extracted into the aqueous layer moved as a single band in Solvent C (Fig. 3B). The polar materials released from GSPL, either by mild alkaline hydrolysis (1 M KOH for 18 hr at 37°C) or by phospholipase C treatment, had identical Rf values on TLC in Solvent C. After about a 15min digestion of the cell surface GSPL with the enzyme, approximately 48% of the original [3H]galactose-labeled material appeared in the aqueous layer (Fig. 4) and this percentage increased with longer exposure to the enzyme, until finally upon prolonged treatment (2 hr) nearly 100% of the original labeled material appeared in the aqueous layer. Treatment with heat-killed enzyme had practically no effect on the hydrolysis of the polar head group of GSPL. The 10% loss of labeled material upon prolonged treatment with heat-killed enzyme might be
ANTIGEN
255
the result of spontaneous hydrolysis under the experimental conditions. The anionic glycolipid bound to the DEAE-Sephadex A-25 column (Figs. 5A and 5C). When [3H]mannose or [32P]phosphate-labeled GSPL was treated with mild alkali followed by alkaline phosphatase digestion and anion-exchange chromatography, no radiolabeled material could be eluted from the column with a KC1 gradient (Figs. 5B and 5D). If GSPL was not subjected to mild alkali treatment prior to enzymatic digestion, the phosphate group remained intact and hence [3H]mannose- or [3H]galactose-labeled material could be eluted from the anion exchanger with a KC1 gradient. Thus, the fact that the phosphate group in GSPL became susceptible to the enzyme only when the glycolipid was mild alkali treated suggests that the phosphate group of GSPL is located internally and is probably attached to the ceramide at the site of the alkali-catalyzed hydrolysis. To establish the presence of the phosphoinositol moiety in GSPL, myo[14C]inositol-labeled GSPL was subjected to acid hydrolysis under controlled conditions (2 N HCl, 1 hr at lOO’C), conditions under which all glycosidic bonds are hydrolyzed with the formation of inositol monophosphate from inositol phosphoceramide. Upon anion-exchange chromatography, 97% of the labeled material could be eluted from the column. When the phosphate group was removed from the eluted material by alkaline phosphatase digestion prior to anion-exchange chromatography, 98% of the labeled material came out unretarded from the column. These data strongly sug‘Time in min. gest that the inositol moiety in GSPL is atFIG. 4. Phospholipase C digestion of parasite lysates. Parasite lysates (5 x 108) metabolically prela- tached to the phosphate group. To establish that the glycolipid was not beled with [3H]galactose were subjected to phospholipase C digestion for diierent lengths of time, and the contaminated by LPG or other GIPLs, puradioactivity in the purified GSPL was determined. rified GSPL was saponified. Under the conThe percentage radioactivity in purified GSPL was de- ditions of saponification (6 N KOH/95% altermined relative to the total radioactivity incorpocohol to a final concentration of 2 N KOH), rated in 5 x 10’ cells. Parasite lysate digested with phosphoinositides phospholipase C from B. cereus (0) and with heat- ceramide-containing give fatty acids only, whereas 1-O alkyl hilled phospholipase C (0).
0
256
TRIF’TI
DE-MAJUMDAR
0.10
6.0 4.0
L.”
0.05
r ,
4005
I/&
20 0.0
10 0 ; 10.0 ,” 8.0 6.0
x E 2
I
I_ u.10
II
/
4.0
005
2.0 12 24 36 Frocfion Number
0.0 48
12 24 36 Fraction Number
46
FIG. 5. Chromatography on DEAE-Sephadex A-25 of the [3ZP]phosphate-and [3H]mannose-labeled glycolipid. The affinity-purified GSPL was applied to a column of DEAE-Sephadex A-25 (0.5 x 5 cm) equilibrated in Solvent D. Fractions of 0.5 ml were collected and measured for radioactivity. After the eighth fraction was collected, a gradient of KC1 (O-O.1 M) in Solvent D was applied to the column. (A) [32P]Phosphate-labeled GSPL; (B) [32P]phosphate-labeled GSPL pretreated with mild alkali and then treated with alkaline phosphatase (0) for 30 min and (0) prolonged treatment; (C) [3H]mannoselabeled GSPL; (D) [3H]mannose-labeled GSPL pretreated with mild alkali and then treated with alkaline phosphatase (prolonged treatment).
glycerol-containing phosphoinositides give alkyl glycerol. Upon saponification, GSPL showed the presence of fatty acid only. The binding of purified GSPL to the antibody raised against the L. donovani whole-cell promastigotes was examined by a RIA (Fig. 6). The binding of the antibody increased with a corresponding increase in the amount of GSPL. The ratio of neutral and amino sugars was determined by GLC on a ECNSS-M column as the amount of reduced and acetylated compounds. Hydrolysis with 2
0
5.0
10.0
15.0 20.0 25.0 30.0 Antigen (1)
35.0 40.0
45.0
FIG. 6. Microassay for GSPL-antibody binding. All negative controls were less than 5000 cpm.
M TFA for 4 hr at 100°C provided mainly mannose and galactose (Table II). On the other hand, hydrolysis with 3 M HCl in methanol for 18 hr at 80°C followed by hydrolysis with 6 M HCl for 18 hr at 105”C, TABLE II Gas Chromatographic Analysis of GSPL
Component Galactose Mannose Inositol 2-Deoxy-2-acetamido-o-glucose Phosphate” C- 16 fatty acid C-18 fatty acid cis-A9-C-18 fatty acid Sphingosineb
Proportion of peak obtained’ (%I 37.09 37.09 4.00 5.06 4.20
4.10 0.78
0.11 6.90
a Phosphate was measured by the method of Bartlett (1959) on the native lipid. b Detected by the spectrophotometric method of Lauter and Trams (1962). c Mean percentage of three individual determinations.
Leishmania donovani ANTIGEN followed by sodium borohydride reduction and acetylation provided mainly glucosamine and myo-inositol (Table II). Non-Nacetylated glucosamine forms glycosidic linkages that are stable under the hydrolysis conditions employed for neutral sugar analysis (2 M TFA for 4 hr at 100°C). The structure of the lipid moiety was analyzed after acid methanolysis as described under Materials and Methods. Sphingolipid bases were quantitated according to the method of Lauter and Trams (1962). Fatty acids were analyzed for their methyl esters and consisted mainly of palmitic acid (74%), stearic acid (14%), and oleic acid (2%) (Table II).
257
man and Goding 1985; Handman et al. 1984; Turco 1988), or by the other abundant GIPLs of the promastigotes. To examine this possibility, GSPL was subjected to saponification. GSPL showed only the presence of fatty acid as derived from the ceramide. The presence of LPG or GIPL contaminant would have resulted in the presence of alkyl glycerols in the H:DE (3: 1; v/v) fraction. To establish the presence of the phosphoinositol moiety, myo-[t4C]inositollabeled GSPL was treated with mild acid under controlled conditions (2 N HCl, v/v; lOO”C/l hr), conditions that allowed the hydrolysis of all glycosidic bonds and formed inositol monophosphate from inositol phosDISCUSSION phoceramide. The anion-exchange chroTreatment of L. donovani promastigotes matographic behavior of [14C]inositol with galactose oxidase (E.C. 1.1.3.9) fol- monophosphate, before and after alkaline lowed by reduction with tritiated sodium phosphatase treatment, established that the borohydride (at pH 7.4) allowed the label- inositol was attached to the phosphate. ing of the GSPL on the external surface of The formation of phosphoinositol olithe cells with tritium, thus indicating the gosaccharide by alkaline hydrolysis (1 M presence of terminal galactose or N-acety- KOH, 18 hr, 37”C), the susceptibility of the lated galactosamine in the GSPL. Further, phosphate group of this oligosaccharide to this glycolipid was found to be anionic in alkaline phosphatase, and the presence of a nature. Hence, the lipids extracted with ceramide in the hexane layer suggest that Solvent A from promastigote homogenates the phosphoinositol group is linked to the were first grossly separated by anion- ceramide. The data obtained by mild alkaexchange chromatography. The anionic line hydrolysis or controlled acid hydrolyglycolipids thus obtained were further sep- sis indicated the presence of inositol(larated according to their polarity on a si- O)phospho( 1-0)ceramide in the GSPL. licit acid adsorption column by employing The release of polar material from the a stepwise elution scheme. The GSPL- parasite surface when it was incubated with enriched fraction (C:M; 4:6; v/v) was sub- phospholipase C from B. cereus could be jected to affinity chromatography on a due to either the action of phospholipase C RCA-Sepharose 4B column. The GSPL or the spontaneous hydrolysis of the GSPL eluted from the column with galactose under the experimental conditions. To exmoved as a single band on TLC in three amine the later possibility, parasite lysates different solvent systems. It was evident (5 x lo8 cells) were subjected to incubation that, following the purification scheme out- at 37°C for varying periods of time in preslined in Fig. 1, the probability existed that ence of the active enzyme and heat-killed this preparation might have been contami- enzyme. The 10% loss of GSPL from the nated by LPG, the major cell surface gly- parasite surface in the absence of the encolipid of all Leishmania species studied so zyme compared to a 100% loss of the glyfar (King et al. 1987; McConville and Bacic colipid in presence of the enzyme argues 1989; McConville et al. 1987, 1990; Hand- against the spontaneous hydrolysis of the
258
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DE-MAJUMDAR
glycolipid. The data obtained by phospho- lated from a patient in Rajasthan). Work on lipase C treatment of L. donovani pro- detailed structural analysis of the GSPLmastigotes or the purified glycolipid sug- like glycolipids is in progress in our laboragest that this glycolipid is anchored in the tory. lipid bilayer via the ceramide moiety. As yet, the biological significance of The neutral sugars and fatty acid compo- GSPL is unknown, although initial experisitions were identified by their characteris- ments suggest that this glycolipid antigen tic GLC retention times. The oligosaccha- can both cross-react with the serum of ride core of the GSPL from L. donovani Kala-azar patients and provide protection consists of a glycosylated phosphoinositol to the parasite upon entry into the phagolystructure, consisting of galactose, man- sosome of a host cell by inhibiting the acnose, inositol, and GlcNH,, and resembles tion of some of the hydrolytic enzymes the carbohydrate core of the LPG of L. (manuscript in preparation). donovani (Turco et al. 1987) and the GIPLs ACKNOWLEDGMENTS isolated from L. major (McConville and Batic 1989). In contrast to the Leishmuniu I am grateful to Professor Amar Bhaduri and Prophosphoinositol-containing lipid types, in- fessor Subhash Basu for their constant encouragement cluding LPG, the GSPL from the L. dono- and valuable guidance throughout the course of this vuni promastigotes contains ceramide as its work. This work was supported by Grant IND/87/018/ lipid moiety, a feature also found in the li- A/O199 from the United Nations Development Programme and the CSIR, India. popeptidophosphoglycan from T. cruzi (Previato et al. 1990), in the lipophosphoREFERENCES glycan from Acunthumoebu custellani (Dearborn et al. 1976), in the glycophos- ALBERSHEIM, P., NEVINS, D. J., ENGLISH, P. D., AND KARR, A. 1967. The analysis of sugars in plant phosphingolipid of T. foetus (Beach et al. cell-wall polysaccharides by gas-liquid chromatog1990), Aspergillus niger (Brennan and Roe raphy. Carbohydrate Research 5, 340-345. 1975; Brennan and Lose1 1978), and To- BARTLETT, G. R. 1959. Phosphorus assay in column bacco leaves (Hsieh et al. 1978), and in the chromatography. Journal of Biological Chemistry 234, 466468. anchor of a protein in D. discoideum (Stadler et al. 1989). The phosphoinositol oli- BASU, M., AND BASU, S. 1973.Enzymatic synthesis of a blood group B-related pentaglycosylceramide by gosaccharide moiety of the T. cruzi lipoan o-galactosyl transferase from rabbit bone marpeptidoglycan contained Man:Gal in a 4:2 row. Journal of Biological Chemistry 248, 1700molar ratio, whereas in the T. fuetus phos1706. phoinositol ceramide-containing lipid, myo- BASU, M., DE, T., DAS, K. K., KYLE, J. W., CHON, H. C., SCHAEPER,R. J., AND BASU, A. 1987. Glyinositol was the only sugar detected. On the colipids. In “Methods in Enzymology” (V. Ginsother hand, the L. donovuni GSPL shows burg, Ed.), Vol. 138, pp. 575407, Academic Press, the presence of Man:Gal in a 1:1 molar raOrlando, FL. tio, in addition to myo-inositol. A similar BEACH, D. H., HOLZ, G. G., SINGH, B. N., AND LINDMARK, D. G. 1990. Fatty acid and sterol meglycolipid has also been isolated from the tabolism of cultured Trichomonas vaginatis and TriL. donovuni strains MOHM/IN/83/Ag 83 trichomonas foetus. Molecular and Biochemical and the WHO reference strain DDB. ReParasitology 38, 175-190. cently, an antibody against the purified BERMAN, J. D., AND DWYER, D. M. 1981. Expression GSPL antigen has been raised in our laboof Leishmania antigen on the surface membrane of infected human macrophages in vitro. Clinical and ratory, and preliminary experiments sugExperimental Immunology 44, 342-348. gest that this antibody does not recognize BRENNAN, P. J., AND LOSEL, D. M. 1978. Physiology either LPG, isolated according to the of fungal lipids: selected topics. Advances in Micromethod of Turco et al. (1987), or GIPL, isobial Physiology 17, 47-179. lated from an Indian strain of L. major (iso- BRENNAN, P. J., AND ROE, J. 1975. The occurance of
Leishmania donovani a phosphorylated glycosphingolipid in Aspergillus niger. The Biochemical Journal 147, 179-180. CHAUDHURY, G., GHOSHAL, K., PAL, S., SEN, S., AND BANERJEE, A. B. 1986. A new medium for large scale production of Leishmania donovani promastigotes for biochemical studies. Indian Journal of Medical Research 84, 451-460. CULLIS, P., AND DE-KRUIFF, B. 1979. Lipid polymorphism and the functional roles of lipids in biological membranes. Biochimica et Biophysics. Acta 559, 3w20. DATTA, A. K., BHAUMIK, D., AND CHATTERIEE, R. 1987. Isolation and characterization of adenosine kinase from Leishmania donovani. Journal of Biological Chemistry 262, 5515-5521. DEARBORN, D. G., SMITH, S., AND KORN, E. D. 1976. Lipophosphoglycan of the plasma membrane of Acanthamoeba castellani. Journal of Biological Chemistry 251, 2976-2982. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. 1956. Calorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350-356. FERGUSON, M. A. J., AND WILLIAMS, A. F. 1988. Cell surface anchoring of proteins via glycosylphosphatidyl inositol structure. Annual Review of Biochemistry 57, 285-320. GHAMBARG, G. G., AND HAKOMORI, S.-I. 1973. External labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes. Journal of Biological Chemistry 248, 43114317. HANDMAN, E., AND GODING, J. W. 1985. The Leishmania receptor for macrophages is a lipid containing glycoconjugate. EMBO Journal 4, 329-336. HANDMAN, E., GREENBLATT, C. L., AND GODING, J. W. 1984. An ampipathic sulphated glycoconjugate of Leishmania: Characterization with monoclonal antibodies. EMBO Journal 3, 2301-2306. HANDMAN, E., MCCONVILLE, M. J., AND GODING, J. W. 1987. Carbohydrate antigens as possible parasite vaccines: A case for Leishmania glycolipid. Immunology Today 8, 181-185. HANDMAN, E., SCHNUR, L. F., SPIT-HILL, T. W., AND MITCHELL, G. F. 1986. Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages. Journal of Immunology 137, 3608-3613. HSIEH, T. C.-Y., LAINE, R. A., AND LESTER, R. L. 1978. Structure of a major glycophosphoceramide from tobacco leaves; PSL-I:2 deoxy-2-acetamodoD-glucopyranosyl(al+4)-D-glucopyranosyl) al+2)myo-inositol-1-0-phosphoceramide. Biochemistry 17, 3575-3581. JAFFE, C. L., PEREZ, M. L., AND SARFSTEIN, R. 1990. Leishmania tropica: Characterization of a lipophosphoglycan-like antigen recognized by species
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specific monoclonal antibody. Experimental Parasitology 70, 12-24. KANESHIRO, E. S., JAYSIMHULU, K., AND LESTER, R. L. 1986. Characterization of inositol lipids from Leishmania donovani promastigotes: Identification of inositol sphingolipids. Journal of Lipid Research 27, 12941303. KATO, M. 1973. Effect of anticord factor antibody on experimental tuberculosis in mice. Infection and Zmmunity 7, 14-21. KING, D. L., CHANG, Y. D., AND TURCO, S. J. 1987. Cell surrface lipophosphoglycan of Leishmania donovani. Molecular and Biochemical Parasitology 24,47-53. LAINE, R. A., AND HSIEH, T. C.-Y. 1987. Inositol containing sphingolipid. In “Methods in Enzymology” (V. Ginsburg, Ed.), Vol. 138, pp. 186-195. Academic Press, Orlando, FL. LAUTER, C. J., AND TRAMS, E. G. 1962. A spectrophotometric determination of sphingosine. Journal of Lipid Research 5, 136138. MCCONVILLE, M. J., AND BACIC, A. 1989. A family of glycoinositol phospholipids from Leishmania major: Isolation, characterization and antigenicity. Journal of Biological Chemistry 264, 757-166. MCCONVILLE, M. J., BACIC, A., MITCHELL, G. F., AND HANDMAN, E. 1987. Lipophosphoglycan of Leishmania major that vaccinates against cutaneous leishmaniasis contains an alkylglycerophosphoinosito1 anchor. Proceedings of the National Academy of Sciences USA 84, 8941-8945. MCCONVILLE, M. J., THOMAS-OATES, J. E., FERGUSON, M. A. J., AND HOMANS, S. W. 1990. Structure of the lipophosphoglycan from Leishmania major. Journal of Biological Chemistry 265, 19,61 l-19,623. ORLANDI, P. A., AND SALVATORO, J. J. 1987. Structure of the lipid moiety of the Leishmania donovani lipophosphoglycan. Journal of Biological Chemistry 262, 10,384-10,391. PREVIATO, J. O., GORIN, P. A. J., MAZUREK, M., XAVIER, M. T., FOURNET, B., WIERUSZESK, J. M., AND MENDONCA-PREVIATO, L. 1990. Primary structure of the oligosaccharide chain of lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi. Journal of Biological Chemistry 265, 25182526. RAY, J. C. 1932. Cultivation of various Leishmania parasites on solid medium. Zndian Journal of Medical Research 20, 355-357. REISSIG, J. L., STROMINGER, J. L., AND LELOIR, L. F. 1955. A modified calorimetric method for the estimation of N-acetylamino sugars. Journal of Biological Chemistry 217, 959-966. ROSEN, G., PAHLSSON, P., LONDNER, W. V., WESTERMAN, M. E., AND NILSSON, D. 1989. Structural analysis of glycosylphosphatidylinositol antigens of
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SARKAR,S., AND BHADURI, A. 1987. Preparation and characterization of ghosts from Leishmania donovani promastigotes. In “Current Trends in Life Sciences: Biomembrane Structure, Biogenesis and Transport” (L. Packer and C. Rajaminckam, Eds.), Vol. 13, pp. 305-310. Today and Tomorrow’s Printers and Publishers, New Delhi (India). SINGH, B. N., CASTELLO,C. E., BEACH, D. H., AND HOLZ, G. G., JR. 1990. Characterization of glycosylphosphatidylinositol lipids of parasitic protozoans: Leishmania mexicana promastigotes, Trypanosoma cruzi Peru epimastigotes and Tritrichomonas foetus. Indian Journal ofBiochemistry and Biophysics 27,411-415. SMITH, S. W., AND LESTER, R. L. 1974. Inositol phosphoryl ceramide, a novel substance and the chief member of a major group of yeast sphingolipids containing a single inositol phosphate. Journal of Biological Chemistry 249, 3395-3405.
STADLER,J., KEENAN, T. W., BAUER, G., AND GERISH, G. 1989. Contact site-A glycoprotein in Dictyostelium discoideum is anchored by a ceramide based lipid glycan. EMBO Journal 8, 371-377. SVENNERHOLM,L. 1958. Quantitative estimation of sialic acid. III. An anion exchange resin method. Acta Chemica Scandenevia 12, 547-554. TURCO, S. J. 1988. The lipophosphoglycan of Leishmania. Parasitology Today 4, 255-257. TURCO, S. J., HULL, S. R., ORLANDI, P. A., AND SHEPHERD,S. D. 1987. Structure of the major carbohydrate fragment of the Leishmania donovani lipophosphoglycan. Biochemistry 26, 6233-6238. YAMAKAWA, T., AND NAGAI, Y. 1978. Glycolipids at the cell surface and their biological functions. Trends in Biochemical Science 3, 128-13 1. WHO 1984.The Leishmaniasis. The Technical Report No. 701. Received June 20, 1991; accepted with revision October 21, 1991
74,251-260(1992)
donovani: Purification and Partial Characterization Glycophosphosphingolipid Antigen Expressed on Promastigote Surface
Leishmania
of a
TRIPTI DE-MAJUMDAR Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta - 700 032, India DE-MAJUMDAR, T. 1992.Leishmania donovani: Purification and partial characterization of a glycophosphosphingolipid antigen expressed on promastigote surface. Experimental Parasitology 74,251-260. A ceramide-anchored glycophosphosphingolipid antigen was isolated from the lipid extract of Leishmania donovani promastigotes. The aflinity-purified glycolipid antigen contained galactose, mannose, myo-inositol, phosphate, ceramide, and hexosamine but no sialic acid. The phosphate group was present internally at the core of the structure: inositol (l-O)phosphorylceramide. The phosphate group became susceptible to alkaline phosphatase only after alkali-catalyzed hydrolysis of the glycolipid. o 1s~ Academic press, IIIC. INDEX DESCRIPTORS AND ABBREVIATIONS: Leishmania donovani; Kala-azar; glycophosphosphingolipid; membrane antigen; ceramide anchor; GIPL, glycophosphatidylinositol lipids; GSPL, glycophosphosphingolipid of Leishmania donovani; RIA, radioimmunoassay; RCA-I, Ricinus communis agglutinin; PMSF, phenylmethylsulfonyl fluoride; H:DE, hexane:diethyl ether; C:M, chloroform:methanol; GlcNH,, glucosamine; GLC, gas-liquid chromatography; TFA, trifluoroacetic acid; PBS, phosphate-buffered saline; Solvent A, chloroform:methanol:ethyl acetate:pyridine:4.5 N ammonia:water (15:15:5:0.5:0.5:0.5, v/v); Solvent B, acetone:diethyl ether:acetic acid (30:70:2, v/v); Solvent C, chloroform: methanol:0.25 N ammonium hydroxide in 0.25% KC1 (65:45:9, v/v); Solvent D, 0.01 M phosphate buffer, pH 6.4, containing 0.05 N ammonium hydroxide and 0.1% sodium salt of taurodeoxycholic acid; Solvent E, pyridine:ethyl acetate:acetic acid:0.25% KC1 (36:36:7:21, v/v); Solvent F, I-butanol:pyridine:0.25% KC1 (3:2:1, v/v).
of antigenic determinants (Yamakawa and Nagai 1978; Cullis and De-Kruijff 1979). A Leishmania donovani is a protozoan par- class of GIPLs, anchored to the cell memasite that causes visceral leishmaniasis or brane via diacyl glycerols, alkyl acyl glycKala-azar, a disease widely prevalent in erols, lysoalkyl glycerols, or ceramides, many parts of the tropical world and gener- has been found in several eukaryotic cells ally fatal if allowed to go untreated (WHO (Ferguson and Williams 1988), including 1984). Little is known about how the para- the parasitic protozoan Leishmania (Handsite is recognized by the host macrophages man et al. 1984; Handman and Goding and how it avoids destruction, although in 1985; King et al. 1987; McConville et al. recent years it is becoming increasingly ev- 1987, 1990; Orlandi and Salvatoro 1987; ident that surface glycoconjugates, includ- Turco et al. 1987; Rosen et al. 1989; Jaffe et ing glycolipids of pathogens, play a crucial al. 1990). The glycophosphosphingolipids conrole in host-parasite interactions or in protection against them (Kato 1973; Berman taining a phosphodiester linkage between and Dwyer 1981; Handman et al. 1986, inositol and ceramide (inositol (l-O)1987; Turco 1988; McConville et al. 1987; phosphoryl-(O-l) ceramide) are a class of GIPLs in which the lipid is anchored to the Rosen et al. 1989). cell membrane via ceramides. These have Lipids, which are essential structural components of biological membranes, play been described mainly in plants, yeasts, and fungi, and it has been suggested that a crucial role in the cell surface recognition, the cell-cell interaction, and the expression these may be functional analogues of the INTRODUCTION
251 OOl4-4894/92$5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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gangliosides found in the animal cell plasma membrane (Laine and Hich 1987). Recently, similar compounds have been reported in the parasitic protozoans Trypanosoma cruzi (Previato et al. 1990) and Tritrichonomonasfoetus (Beach et al. 1990) and also in the slime mold Dictyostelium discoideum (Stadler et al. 1989), but to date, no GSPLs have been reported from any of the Leishmania species studied so far. A ceramide-anchored inositol sphingolipid has been reported from L. donovani, but it is not glycosylated (Kaneshiro et al. 1986). This paper reports the purification and partial characterization of a ceramideanchored GIPL antigen from the L. donovani promastigote surface membrane. The potential biological role of the antigen is discussed. MATERIALS
AND METHODS
Leishmania parasites. Leishmania strain UR6 (MHOIIN/1978/UR6) was isolated from an Indian patient with Kala-azar (Datta et al. 1987). The organisms were maintained and grown in modified Ray’s medium (Ray 1932) and subcultures were made at 72-hr intervals. For radiolabeling studies, parasites were grown in the semidefined medium of Chaudhury et al. (1986). Preparation of antisera. Antibodies against the whole-cell promastigote homogenates were prepared by four subcutaneous injections into rabbits (2 x IO6 promastigotes). Samples of the extracted glycolipid were tested for antigenicity by a RIA, according to the method of Basu et al. (1987) and 2-18 ng of antigen was taken in each well. Lacto-neo-pentaglycosylceramide was used as a negative control (Basu and Basu 1973). Metabolic labeling ofparasites. Late log phase promastigotes (2 x 108) from blood agar cultures were washed three times with PBS and were biosynthetitally labeled with 500 pCi [32P]orthophosphate in 5 ml of a phosphate-free medium of Chaudhury et al. (1986), with 100 pCi of [‘Hlmannose or [3H]galactose in 5 ml of a glucose-free medium (Chaudhury et al. 1986), with 10 nCi of myo-[U-‘4C]inositol, with 10 nCi of [l-‘4C]palmitic acid, or with 100 nCi of D-[6-3H]GlcNH,, for 3 hr at room temperature. External labeling of the cell surface carbohydrates was done according to the method of Ghambarg and Hakomori (1973). Radiolabeled parasites were collected, washed in PBS, and extracted with Solvent A, and the glycolipid was purified as described in the text.
Preparation of a promastigote membrane devoid of internal material and jlagella. Ghosts from L. donovani promastigotes were prepared according to a
method developed in our laboratory (Sarkar and Bhaduri 1987). Briefly, late log phase (1 g wet wt) flagellated promastigotes were taken up in 50 mM Tris-HCl, pH 7.4, containing 2 mM PMSF and were kept on ice for 2 hr with occasional vortexing. Cells were collected by centrifugation and the pellet was washed extensively with PBS. The partially purified membrane fraction from a discontinuous sucrose density gradient centrifugation [5%(2 m1)/25%(2 ml), w/v] was further purified on a second density gradient. This purified preparation was used throughout. Extraction and purification of glycophosphosphingolipid. Late log phase promastigotes (1 g wet wt)
were washed with PBS and extracted with 19 vol of Solvent A. The extracted material was concentrated by rotary evaporation and loaded onto a column of DEAE-Sephadex A-25 (0.5 x 5 cm) equilibrated in Solvent D. The column was washed with 5 ml of the solvent, and the anionic glycolipids were eluted with a gradient of KC1 in Solvent D. The fraction containing the GSPL antigen was precipitated with 1 vol of acetone. After 3 days at 4°C to allow the precipitate to settle, the supernatant was siphoned off. The precipitate was washed with acetone and dried. The dried precipitate was taken up in 1 ml of C:M (9: 1, v/v) and loaded onto a silicic acid column preequilibrated in chloroform (10 g silicic acid/g lipid). The column was sequentially eluted with 10 bed vol each of chloroform and then with C:M (8:2; 7:3; 6:4; 1:l; 4:6; 3:7; 2:8; v/v) and finally with methanol. The fraction containing the GSPL antigen (C:M; 4:6; v/v) was further purified on a RCA-I-Sepharose 4B affinity column (0.8 x 8 cm, equilibrated in Solvent D). The column was washed with 10 bed vol of Solvent D. and the adsorbed material was eluted with 5 ml of 0.1 M galactose in Solvent D. The galactose eluent was dialyzed against PBS at 4°C for 8 hr with frequent changes of buffer and finally lyophilized. Each fraction was monitored for the presence of the glycolipid antigen by TLC in Solvent C and autoradiography. Acid hydrolysis. After acid hydrolysis of GSPL, neutral sugar was estimated by the phenol-sulfuric acid method of Dubois et al. (1956). Hexosamine was estimated according to the method of Reissing et a/. (1955) and sialic acid according to the method of Svennerholm (1958). Phosphorus was assayed by the Bartlett (1959) method, and long chain bases were quantitated according to the method of Lauter and Trams (1962) after acidic methanolysis of GSPL. Sphingosine was used as standard. Anion-exchange chromatography. The purified GSPL was subjected to anion-exchange chromatography on a DEAE-Sephadex A-25 column (0.5 x 5 cm), and the bound material eluted with a linear gradient of
Leishmania donovani ANTIGEN KC1 (O-O.1it4) in Solvent D. Fractions of 500 ~1 were collected and counted for radioactivity in a liquid scintillation counter. The GSPL was also digested with alkaline phosphatase, before and after treatment with mild alkali (1 M KOH, 18 hr, 37°C) and subjected to anion-exchange chromatography as described for the untreated GSPL. Mild acid hydrolysis under controlled conditions followed by anion-exchange chromatography. Mild acid
hydrolysis of the myo-[i4C]inositol-labeled GSPL was carried out under controlled conditions. GSPL (18320 cpm/lOO pl) was treated with 2 N HCl(100 ~1)for 1 hr at 100°C. After removal of HCl, the hydrolyzate was subjected to anion-exchange chromatography on a DEAE-Sephadex A-25 column (0.5 x 5 cm), and the material eluted with a linear gradient of KC1 (O-O.1 W in Solvent D. The eluted material was further subjected to anion-exchange chromatography under identical conditions after treatment with alkaline phosphatase. Mild alkali treatment. Mild alkali treatment of GSPL was carried out with 1 M KOH, for 18 hr at 37°C (Smith and Lester 1974). After neutralization with acetic acid, the nonpolar material was extracted with hexane. The aqueous layer was desalted on a Bio-Gel P-2 column (1.0 x 50 cm), and the material eluted in the void volume was subjected to TLC in Solvent C and exposed to autoradiography. The eluted material was also analyzed before and after treatment with alkaline phosphatase for the presence of phosphorus, galactose, mannose, GlcNH,, and inositol. Saponification ofGSPL. GSPL (1 mg) was refluxed for 8 hr in 10 ml of a mixture of acetic acid:acetic anhydride (3:2). After saponification in 6 N KOH in 95% alcohol to a final concentration of 2 N KOH, the glycosides and other nonsaponifiable products were removed by ether extraction. The saponified mixture was loaded onto a silicic acid column and after the fatty acid was eluted with H:DE (3:1), the alkyl glycerols were removed by H:DE (1:3). Both the eluents were subjected to TLC on silica gel G in Solvent B and visualized by H,SO, charring. Phospholipase C treatment. Purified GSPL (9.96 X lo5 cpm of [3H]galactose, 10.44 x lo5 cpm of [3H]mannose, or 10.34 X lo4 cpm of [14C]palmitate) was incubated with phospholipase C from Bacillus cereus (0.05 p&ml) in 50 pl of 25 mM Hepes, pH 7.5, for 1 hr at 37°C. Polar materials were extracted with water, subjected to TLC in Solvent C, and exposed to autoradiography. The digestion of the cell surface GSPL by the enzyme was monitored by using [3H]galactose-labeled parasite lysate. Parasite lysates (5 x 10scells, 1 ml, 25 mM Hepes, pH 7.5) were subjected to enzymatic digestion (0.05 &ml of enzyme) for varying periods of time and after the cells were washed free of the enzyme, the GSPL was purified as described in the text. Enzyme boiled for 5 min was used as a negative control.
253
GLC analysis of neutral sugar. After hydrolysis of samples with 2 M TFA for 4 hr at lOO”C, the neutral sugars were converted to their alditol acetates (Albersheim et al. 1%7) and analyzed by GLC on a 6-ft column of 3% ECNSS-M with a temperature program of 2Wmin from 160 to 250°C at a flow rate of 20 ml/min of carrier gas. GLC analysis of inositol and amino sugars. Inositol and GlcNH, present in the GSPL were analyzed by GLC after hydrolysis with 3 M HCl in methanol for 18 hr at 80°C. The methanolysates were dried under a stream of nitrogen, and the resulting material was dissolved in 1.0 ml of aqueous 6 M HCl and heated at 105°C for 18 hr. Fatty acids were extracted with hexame and the aqueous layer was lyophilized. The lyophilized residue was reduced with sodium borohydride and acetylated with acetic anhydride-pyridine (Albersheim et al. 1967), and the products were analyzed by GLC as described for neutral sugar analysis. GLC analysis of fatty acids. GSPL (500 kg) was methanolyzed with 0.5 M HCI in methanol (1 ml) for 18 hr at 80°C. After hexane extraction, the hexane layer was analyzed for the fatty acid methyl esters by GLC on a 6ft column of 3% ECNSS-M with a temperature program of 2”Clmin from 135to 202°C at a flow rate of 25 ml/min carrier N, gas.
RESULTS
Approximately 1 g (wet wt) of L. donovani promastigotes was extracted exhaustively with Solvent A (Fig. 1). The extracted glycolipids were fractionated on an anion exchanger, and the anionic lipids thus obtained were further separated on a silicic acid column according to their polarity. Three phosphoinositol lipid-containing fractions were obtained. The C:M (4:6) fraction showed the presence of GSPL antigen along with a closely moving band. The other two fractions showed the presence of glyceryl ethers. The GSPL antigen finally bound to the RCA-I-Sepharose affinity column and was selectively eluted with 0.1 M galactose in Solvent D. During the purification process, each fraction was monitored by TLC and visualized by exposing the TLC plates to I, vapor. Purified GSPL moved as a single band in three different solvent systems (Solvents C, E, and F) (Fig. 2). This material could be metabolically labeled with GlcNH,, galactose, man-
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DE-MAJUMDAR
Late log phase promastigotes 1 Promastigote ghosts Extraction with 19 vol of I Solvent A DEAE-Sephadex A-25 column (0.5 x 5 cm) O-O.1 M KC1 gradient I GSPL containing fraction precipitated with acetone I Precipitate dissolved in C:M (9: 1; v/v) 1 Silicic acid column C:M (4:6; v/v)
I Lectin-Sepharose affinity column (0.8 x 9 cm) 0.1 M galactose in I Solvent D(5 ml) Purified GSPL FIG. 1. Scheme of extraction and purification of the glycolipid.
nose, myo-inositol, palmitic acid, and phosphate. The GSPL could also be surface labeled by the galactose oxidase/NaB3H, procedure (Table I) (Ghamberg and Hakomori 1973). To establish the linkage of the phosphoinositol group to the ceramide, GSPL was subjected to mild aqueous alkaline hydrolysis under controlled conditions (1 M
4 2 3 1 FIG. 2. TLC of the radiolabeled purified GSPL. Lane 1, [3H]galactose; Lane 2, [3H]mannose; Lane 3, ‘*P-labeled affinity-purified GSPL antigen; Lane 4, 32P-labeled C:M (4:6) fraction from silicic acid column (20,000 cpm of 3H- and 500 cpm of “P-labeled compound). Chromatograms were developed in Solvent C and subjected to autoradiography.
TABLE I Metabolic and Surface Labeling Studies of L. donovani promastigotes
Metabolite Mannose Galactose Inositol Palmitic acid Phosphate Galactose oxidase/NaB3H,
Percentage of radioactivity” incorporated into purified GSPL 2.0 2.3 2.1 4.6 0.35 0.5
’ Mean percentage incorporation of radioactivity of three individual determinations.
KOH for 18 hr at 37”C), conditions that would hydrolyze inositol phosphoceramide, with the formation of a cyclic inosito1 phosphate intermediate, finally producing an inositol monophosphate and ceramide (Smith and Lester 1974). On methanolysis, the hexane layer showed the presence of fatty acid and sphingosine. The aqueous layer moved as a single band on TLC in Solvent C (Fig. 3A) and showed the presence of galactose, mannose, inositol, GlcNH,, and phosphate. These results suggest that mild alkali treatment cleaves the ceramide moiety of GSPL, with the release of the corresponding phosphoinositol oligosaccharide in the aqueous layer.
FIG. 3. TLC of the phosphoinositol oligosaccharide obtained from GSPL. (A) [3H]galactose-labeled oligosaccharide obtained by mild alkaline hydrolysis of GSPL, (B) [3H]galactose-labeled oligosaccharide obtained by phospholipase C treatment of GSPL. Chromatograms were developed in Solvent C and subjected to autoradiography.
Leishmania
donovani
The effect of phospholipase C (B. cereus) on both the purified and the membranebound GSPL was examined. The polar material extracted into the aqueous layer moved as a single band in Solvent C (Fig. 3B). The polar materials released from GSPL, either by mild alkaline hydrolysis (1 M KOH for 18 hr at 37°C) or by phospholipase C treatment, had identical Rf values on TLC in Solvent C. After about a 15min digestion of the cell surface GSPL with the enzyme, approximately 48% of the original [3H]galactose-labeled material appeared in the aqueous layer (Fig. 4) and this percentage increased with longer exposure to the enzyme, until finally upon prolonged treatment (2 hr) nearly 100% of the original labeled material appeared in the aqueous layer. Treatment with heat-killed enzyme had practically no effect on the hydrolysis of the polar head group of GSPL. The 10% loss of labeled material upon prolonged treatment with heat-killed enzyme might be
ANTIGEN
255
the result of spontaneous hydrolysis under the experimental conditions. The anionic glycolipid bound to the DEAE-Sephadex A-25 column (Figs. 5A and 5C). When [3H]mannose or [32P]phosphate-labeled GSPL was treated with mild alkali followed by alkaline phosphatase digestion and anion-exchange chromatography, no radiolabeled material could be eluted from the column with a KC1 gradient (Figs. 5B and 5D). If GSPL was not subjected to mild alkali treatment prior to enzymatic digestion, the phosphate group remained intact and hence [3H]mannose- or [3H]galactose-labeled material could be eluted from the anion exchanger with a KC1 gradient. Thus, the fact that the phosphate group in GSPL became susceptible to the enzyme only when the glycolipid was mild alkali treated suggests that the phosphate group of GSPL is located internally and is probably attached to the ceramide at the site of the alkali-catalyzed hydrolysis. To establish the presence of the phosphoinositol moiety in GSPL, myo[14C]inositol-labeled GSPL was subjected to acid hydrolysis under controlled conditions (2 N HCl, 1 hr at lOO’C), conditions under which all glycosidic bonds are hydrolyzed with the formation of inositol monophosphate from inositol phosphoceramide. Upon anion-exchange chromatography, 97% of the labeled material could be eluted from the column. When the phosphate group was removed from the eluted material by alkaline phosphatase digestion prior to anion-exchange chromatography, 98% of the labeled material came out unretarded from the column. These data strongly sug‘Time in min. gest that the inositol moiety in GSPL is atFIG. 4. Phospholipase C digestion of parasite lysates. Parasite lysates (5 x 108) metabolically prela- tached to the phosphate group. To establish that the glycolipid was not beled with [3H]galactose were subjected to phospholipase C digestion for diierent lengths of time, and the contaminated by LPG or other GIPLs, puradioactivity in the purified GSPL was determined. rified GSPL was saponified. Under the conThe percentage radioactivity in purified GSPL was de- ditions of saponification (6 N KOH/95% altermined relative to the total radioactivity incorpocohol to a final concentration of 2 N KOH), rated in 5 x 10’ cells. Parasite lysate digested with phosphoinositides phospholipase C from B. cereus (0) and with heat- ceramide-containing give fatty acids only, whereas 1-O alkyl hilled phospholipase C (0).
0
256
TRIF’TI
DE-MAJUMDAR
0.10
6.0 4.0
L.”
0.05
r ,
4005
I/&
20 0.0
10 0 ; 10.0 ,” 8.0 6.0
x E 2
I
I_ u.10
II
/
4.0
005
2.0 12 24 36 Frocfion Number
0.0 48
12 24 36 Fraction Number
46
FIG. 5. Chromatography on DEAE-Sephadex A-25 of the [3ZP]phosphate-and [3H]mannose-labeled glycolipid. The affinity-purified GSPL was applied to a column of DEAE-Sephadex A-25 (0.5 x 5 cm) equilibrated in Solvent D. Fractions of 0.5 ml were collected and measured for radioactivity. After the eighth fraction was collected, a gradient of KC1 (O-O.1 M) in Solvent D was applied to the column. (A) [32P]Phosphate-labeled GSPL; (B) [32P]phosphate-labeled GSPL pretreated with mild alkali and then treated with alkaline phosphatase (0) for 30 min and (0) prolonged treatment; (C) [3H]mannoselabeled GSPL; (D) [3H]mannose-labeled GSPL pretreated with mild alkali and then treated with alkaline phosphatase (prolonged treatment).
glycerol-containing phosphoinositides give alkyl glycerol. Upon saponification, GSPL showed the presence of fatty acid only. The binding of purified GSPL to the antibody raised against the L. donovani whole-cell promastigotes was examined by a RIA (Fig. 6). The binding of the antibody increased with a corresponding increase in the amount of GSPL. The ratio of neutral and amino sugars was determined by GLC on a ECNSS-M column as the amount of reduced and acetylated compounds. Hydrolysis with 2
0
5.0
10.0
15.0 20.0 25.0 30.0 Antigen (1)
35.0 40.0
45.0
FIG. 6. Microassay for GSPL-antibody binding. All negative controls were less than 5000 cpm.
M TFA for 4 hr at 100°C provided mainly mannose and galactose (Table II). On the other hand, hydrolysis with 3 M HCl in methanol for 18 hr at 80°C followed by hydrolysis with 6 M HCl for 18 hr at 105”C, TABLE II Gas Chromatographic Analysis of GSPL
Component Galactose Mannose Inositol 2-Deoxy-2-acetamido-o-glucose Phosphate” C- 16 fatty acid C-18 fatty acid cis-A9-C-18 fatty acid Sphingosineb
Proportion of peak obtained’ (%I 37.09 37.09 4.00 5.06 4.20
4.10 0.78
0.11 6.90
a Phosphate was measured by the method of Bartlett (1959) on the native lipid. b Detected by the spectrophotometric method of Lauter and Trams (1962). c Mean percentage of three individual determinations.
Leishmania donovani ANTIGEN followed by sodium borohydride reduction and acetylation provided mainly glucosamine and myo-inositol (Table II). Non-Nacetylated glucosamine forms glycosidic linkages that are stable under the hydrolysis conditions employed for neutral sugar analysis (2 M TFA for 4 hr at 100°C). The structure of the lipid moiety was analyzed after acid methanolysis as described under Materials and Methods. Sphingolipid bases were quantitated according to the method of Lauter and Trams (1962). Fatty acids were analyzed for their methyl esters and consisted mainly of palmitic acid (74%), stearic acid (14%), and oleic acid (2%) (Table II).
257
man and Goding 1985; Handman et al. 1984; Turco 1988), or by the other abundant GIPLs of the promastigotes. To examine this possibility, GSPL was subjected to saponification. GSPL showed only the presence of fatty acid as derived from the ceramide. The presence of LPG or GIPL contaminant would have resulted in the presence of alkyl glycerols in the H:DE (3: 1; v/v) fraction. To establish the presence of the phosphoinositol moiety, myo-[t4C]inositollabeled GSPL was treated with mild acid under controlled conditions (2 N HCl, v/v; lOO”C/l hr), conditions that allowed the hydrolysis of all glycosidic bonds and formed inositol monophosphate from inositol phosDISCUSSION phoceramide. The anion-exchange chroTreatment of L. donovani promastigotes matographic behavior of [14C]inositol with galactose oxidase (E.C. 1.1.3.9) fol- monophosphate, before and after alkaline lowed by reduction with tritiated sodium phosphatase treatment, established that the borohydride (at pH 7.4) allowed the label- inositol was attached to the phosphate. ing of the GSPL on the external surface of The formation of phosphoinositol olithe cells with tritium, thus indicating the gosaccharide by alkaline hydrolysis (1 M presence of terminal galactose or N-acety- KOH, 18 hr, 37”C), the susceptibility of the lated galactosamine in the GSPL. Further, phosphate group of this oligosaccharide to this glycolipid was found to be anionic in alkaline phosphatase, and the presence of a nature. Hence, the lipids extracted with ceramide in the hexane layer suggest that Solvent A from promastigote homogenates the phosphoinositol group is linked to the were first grossly separated by anion- ceramide. The data obtained by mild alkaexchange chromatography. The anionic line hydrolysis or controlled acid hydrolyglycolipids thus obtained were further sep- sis indicated the presence of inositol(larated according to their polarity on a si- O)phospho( 1-0)ceramide in the GSPL. licit acid adsorption column by employing The release of polar material from the a stepwise elution scheme. The GSPL- parasite surface when it was incubated with enriched fraction (C:M; 4:6; v/v) was sub- phospholipase C from B. cereus could be jected to affinity chromatography on a due to either the action of phospholipase C RCA-Sepharose 4B column. The GSPL or the spontaneous hydrolysis of the GSPL eluted from the column with galactose under the experimental conditions. To exmoved as a single band on TLC in three amine the later possibility, parasite lysates different solvent systems. It was evident (5 x lo8 cells) were subjected to incubation that, following the purification scheme out- at 37°C for varying periods of time in preslined in Fig. 1, the probability existed that ence of the active enzyme and heat-killed this preparation might have been contami- enzyme. The 10% loss of GSPL from the nated by LPG, the major cell surface gly- parasite surface in the absence of the encolipid of all Leishmania species studied so zyme compared to a 100% loss of the glyfar (King et al. 1987; McConville and Bacic colipid in presence of the enzyme argues 1989; McConville et al. 1987, 1990; Hand- against the spontaneous hydrolysis of the
258
TRIPTI
DE-MAJUMDAR
glycolipid. The data obtained by phospho- lated from a patient in Rajasthan). Work on lipase C treatment of L. donovani pro- detailed structural analysis of the GSPLmastigotes or the purified glycolipid sug- like glycolipids is in progress in our laboragest that this glycolipid is anchored in the tory. lipid bilayer via the ceramide moiety. As yet, the biological significance of The neutral sugars and fatty acid compo- GSPL is unknown, although initial experisitions were identified by their characteris- ments suggest that this glycolipid antigen tic GLC retention times. The oligosaccha- can both cross-react with the serum of ride core of the GSPL from L. donovani Kala-azar patients and provide protection consists of a glycosylated phosphoinositol to the parasite upon entry into the phagolystructure, consisting of galactose, man- sosome of a host cell by inhibiting the acnose, inositol, and GlcNH,, and resembles tion of some of the hydrolytic enzymes the carbohydrate core of the LPG of L. (manuscript in preparation). donovani (Turco et al. 1987) and the GIPLs ACKNOWLEDGMENTS isolated from L. major (McConville and Batic 1989). In contrast to the Leishmuniu I am grateful to Professor Amar Bhaduri and Prophosphoinositol-containing lipid types, in- fessor Subhash Basu for their constant encouragement cluding LPG, the GSPL from the L. dono- and valuable guidance throughout the course of this vuni promastigotes contains ceramide as its work. This work was supported by Grant IND/87/018/ lipid moiety, a feature also found in the li- A/O199 from the United Nations Development Programme and the CSIR, India. popeptidophosphoglycan from T. cruzi (Previato et al. 1990), in the lipophosphoREFERENCES glycan from Acunthumoebu custellani (Dearborn et al. 1976), in the glycophos- ALBERSHEIM, P., NEVINS, D. J., ENGLISH, P. D., AND KARR, A. 1967. The analysis of sugars in plant phosphingolipid of T. foetus (Beach et al. cell-wall polysaccharides by gas-liquid chromatog1990), Aspergillus niger (Brennan and Roe raphy. Carbohydrate Research 5, 340-345. 1975; Brennan and Lose1 1978), and To- BARTLETT, G. R. 1959. Phosphorus assay in column bacco leaves (Hsieh et al. 1978), and in the chromatography. Journal of Biological Chemistry 234, 466468. anchor of a protein in D. discoideum (Stadler et al. 1989). The phosphoinositol oli- BASU, M., AND BASU, S. 1973.Enzymatic synthesis of a blood group B-related pentaglycosylceramide by gosaccharide moiety of the T. cruzi lipoan o-galactosyl transferase from rabbit bone marpeptidoglycan contained Man:Gal in a 4:2 row. Journal of Biological Chemistry 248, 1700molar ratio, whereas in the T. fuetus phos1706. phoinositol ceramide-containing lipid, myo- BASU, M., DE, T., DAS, K. K., KYLE, J. W., CHON, H. C., SCHAEPER,R. J., AND BASU, A. 1987. Glyinositol was the only sugar detected. On the colipids. In “Methods in Enzymology” (V. Ginsother hand, the L. donovuni GSPL shows burg, Ed.), Vol. 138, pp. 575407, Academic Press, the presence of Man:Gal in a 1:1 molar raOrlando, FL. tio, in addition to myo-inositol. A similar BEACH, D. H., HOLZ, G. G., SINGH, B. N., AND LINDMARK, D. G. 1990. Fatty acid and sterol meglycolipid has also been isolated from the tabolism of cultured Trichomonas vaginatis and TriL. donovuni strains MOHM/IN/83/Ag 83 trichomonas foetus. Molecular and Biochemical and the WHO reference strain DDB. ReParasitology 38, 175-190. cently, an antibody against the purified BERMAN, J. D., AND DWYER, D. M. 1981. Expression GSPL antigen has been raised in our laboof Leishmania antigen on the surface membrane of infected human macrophages in vitro. Clinical and ratory, and preliminary experiments sugExperimental Immunology 44, 342-348. gest that this antibody does not recognize BRENNAN, P. J., AND LOSEL, D. M. 1978. Physiology either LPG, isolated according to the of fungal lipids: selected topics. Advances in Micromethod of Turco et al. (1987), or GIPL, isobial Physiology 17, 47-179. lated from an Indian strain of L. major (iso- BRENNAN, P. J., AND ROE, J. 1975. The occurance of
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