surface antigens of leishmania mexicana amastigotes

INTRODUCTION

Protozoa of the genus

Leishmania cause a number of importantdiseases in humans. The parasites undergo a life cycle withthree developmental stages: the flagellated procyclic and meta-cyclic promastigotes, which occur in the digestive tract of thesandfly vector, and the non-motile amastigotes which resideintracellularly in the acidic phagolysosomal compartment ofmammalian macrophages. For each stage, the molecular prop-erties of the cell surface are important for the interaction withhost cells and for protection against hydrolytic enzymes in thesandfly’s gut and in phagolysosomes. Promastigotes containthree abundant surface components, which have been exten-sively investigated: a complex glycoconjugate, termedlipophosphoglycan (LPG) which forms a dense glycocalyx, themetalloprotease gp63 and low molecular mass glycoinositol-phospholipids (GIPLs; see reviews by Chang et al., 1990;McConville, 1991; Turco and Descoteaux, 1992; Medina-Acosta et al., 1993; McConville and Ferguson, 1993). In themammalian host, LPG is considered to protect the parasitesagainst complement-mediated lysis (Puentes et al., 1990) andto serve as a receptor for binding to macrophages either

directly or after fixation of complement (Kelleher et al., 1992).In the insect, the glycoconjugate is important for protectionagainst attack by hydrolases and for attachment to epithelialcells in the midgut (Pimenta et al., 1992).

In comparison to promastigotes, little is known aboutsurface molecules of amastigotes. Expression of LPG isstrongly reduced or undetectable (McConville and Blackwell,1991; Glaser et al., 1991; Turco and Sacks, 1991; Moody etal., 1993; Bahr et al., 1993; Schneider et al., 1993), which cor-relates with the absence of a glycocalyx at the amastigotesurface (Pimenta et al., 1991). Likewise, the metalloproteaseof promastigotes is not expressed in L. major amastigotes(Schneider et al., 1992). L. mexicana amastigotes mainly syn-thesize a soluble isoform of the protease in reduced amounts,which is located in the lumen of lysosomes (Medina-Acosta etal., 1989; Bahr et al., 1993; Ilg et al., 1993). In contrast,amastigotes like promastigotes contain large amounts of GIPLs(~107 molecules/cell). Three distinct types of GIPLs have beendescribed, which differ in their glycan moieties. In L. major,GIPLs of both stages are related to the anchor of LPG (type-2) (McConville et al., 1990a; Schneider et al., 1993). In L.donovani, promastigotes and amastigotes express GIPLs that

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Amastigotes of the protozoan parasite

Leishmania prolif-erate in phagolysosomes of macrophages. They abundantlyexpress glycoinositol phospholipids (GIPLs), which areconsidered necessary for parasite survival by providing ashield at the surface against lysosomal hydrolases and byserving as receptors for the interaction with host cells. Thestructures of four GIPLs of L. mexicana amastigotes werecharacterized by a combination of gas-liquid chromatog-raphy-mass spectrometry, methylation linkage analysisand enzymatic treatments. They contain the glycan struc-tures Man

α1-3Manα1-4GlcN (iM2), Manα1-6(Manα1-3)Manα1-4GlcN (iM3), Manα1-2Manα1-6(Manα1-3)-Manα1-4GlcN (iM4) and (NH2-CH2CH2-PO4)Manα1-6(Manα1-3)Manα1-4GlcN (EPiM3), which are linked toalkylacyl-phosphatidylinositol. The predominant amastig-ote GIPL, EPiM3 (~2×107 molecules/cell), is located at the

parasite cell surface, in the flagellar pocket and inlysosomal membranes, but not on host cell structures asshown by immunofluorescence and immunoelectronmicroscopy. In addition, amastigotes in infected Balb/cmice contain a glycolipid with similar distribution asEPiM3, which has the same characteristics as the Forssmanantigen of mammalian cells. In contrast to EPiM3, there isstrong evidence that this glycosphingolipid is not synthe-sized by amastigotes but by macrophages in the lesion. Thissuggests a mechanism of lipid transfer from themacrophage to the parasite.

Key words: Leishmania mexicana, amastigote, glycoinositol-phospholipid, Forssman glycosphingolipid, macrophage,immunoelectron microscopy

SUMMARY

Surface antigens of

Leishmania mexicana amastigotes: characterization of

glycoinositol phospholipids and a macrophage-derived glycosphingolipid

Gerhard Winter1, Manuela Fuchs1, Malcolm J. McConville2,*,York-Dieter Stierhof1 and Peter Overath1,†

1Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, D72076 Tübingen, Federal Republic of Germany2Department of Biochemistry, University of Dundee, Dundee DDI 4HN, UK

*Present address: Department of Biochemistry, University of Melbourne, Parkville 3052, Australia†Author for correspondence

Journal of Cell Science 107, 2471-2482 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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are homologous to the glycolipid anchors of proteins (type-1)or share features with both the LPG and protein anchors(hybrid type GIPLs) (McConville and Blackwell, 1991).Recently, the structure of the GIPLs of L. mexicana pro-mastigotes has been elucidated, and consists of both hybridtype and lower amounts of the type-2 GIPLs (McConville etal., 1993). A fraction of the hybrid type GIPLs in L. mexicanapromastigotes contain a novel modification at the glucosamineresidue by ethanolamine-phosphate. In the case of L. majorpromastigotes and amastigotes, surface labeling techniqueshave provided evidence that GIPLs are expressed in high copynumber at the cell surface (McConville and Bacic, 1990;Schneider et al., 1993). As major components of the surface ofamastigotes, GIPLs may be of central importance for survivalby shielding the parasite against attack by lysosomal hydro-lases or in mediating host-parasite interactions such as attach-ment to macrophages (Blackwell et al., 1985).

This report shows that L. mexicana amastigotes synthesizethe same GIPLs as promastigotes although in different relativeamounts. In addition, amastigotes isolated from mouse lesionscontain host-derived Forssman antigen (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc1-Ceramide) (Siddiqui andHakomori, 1971). The predominant amastigote GIPL andForssman antigen are located at the surface, in the flagellarpocket and in lysosomal membranes of amastigotes.

MATERIALS AND METHODS

ParasitesPromastigotes of Leishmania mexicana mexicana (strainMNYC/BZ/62/M379) were grown as described (Menz et al., 1991).Amastigotes were isolated from dorsal lesions of Balb/c mice (Hartet al., 1981; Bahr et al., 1993). Alternatively, lesion-derived amastig-otes were cultivated in Schneider’s Drosophila medium, pH 5.5, sup-plemented with 20% inactivated fetal bovine serum (iFCS) at 34°Cin 5% CO2 in air (Bates et al., 1992). They were used after 10 daysof cultivation for the infection of peritoneal macrophages as previ-ously described (Stierhof et al., 1991). Lesions for the cryostatsections and electron microscopy were taken from Balb/c mice 3-5weeks after infection.

Extraction and purification of glycolipidsLipids were prepared as described by McConville et al. (1987). Cellpellets were extracted twice in chloroform/methanol/water (1:2:0.8,v/v) and the insoluble material was removed by centrifugation (12,000g, 10 minutes). Water was added to chloroform/methanol/water-extracts of amastigotes and promastigotes to a final ratio of 4:8:5.6(v/v) (McConville and Bacic, 1989). The upper methanol/water phasewas retained after centrifugation and the lower organic phase waswashed twice with 0.83 volumes of methanol/0.01 M KCl (3:2,v/v).Under these conditions, most of the L. mexicana glycolipids parti-tioned into the upper methanol/water phase. The combined upperphases were evaporated to dryness under a stream of nitrogen, resus-pended in 0.1 M ammonium acetate, pH 6.5, containing 5% 1-propanol, and loaded onto a column of octyl-Sepharose CL-4B (3 cm× 1 cm) equilibrated in the same buffer. After washing with this buffer(20 ml), the column was eluted with 0.1 M ammonium acetate, 40%1-propanol, pH 6.5 (10 ml). The elution of glycolipids was monitoredby high performance thin layer chromatography (HPTLC) analysis ofaliquots from each fraction. Glycolipid-containing fractions werepooled and dried under a stream of nitrogen. Alternatively, glycol-ipids in the chloroform/methanol/water extract were dried and parti-tioned between a lower organic and an upper methanol/water phase

as described by Folch et al. (1957). Dried extracts of 1×109 cells weredissolved in 1.5 ml chloroform/methanol/0.01 M KCl (8:4:3, v/v) andthe two phases separated by centrifugation. The upper methanol/waterphase was removed and the lower organic phase re-extracted oncewith 600 µl methanol/0.01 M KCl (1:1, v/v). Under these conditionsthe GIPLs partitioned into the lower organic phase or precipitated atthe interface of the two phases. Individual glycolipid species werepurified by HPTLC on Silica Gel 60 sheets (Merck, Darmstadt, FRG)using chloroform/methanol/18 M NH4OH/1 M ammoniumacetate/water (180:140:9:9:23, v/v) as the mobile phase. Glycolipidswere located by staining a reference sheet with an orcinol-H2SO4spray reagent and recovered from silica scrapings with four extrac-tions in 5 ml chloroform/methanol/water (1:2:0.8, v/v) for 1-2 hours.

Analysis of GIPLs by Dionex HPLCGlycolipids were treated with phosphatidylinositol-specific phospho-lipase C, deaminated with nitrous acid and subsequently reduced withNaB3H4. Labeled GIPL glycans were then analysed by Dionex HPLCas described by McConville et al. (1993) using an initial lineargradient from 95% buffer A (0.15 M NaOH), 5% buffer B (0.15 MNaOH, 0.25 M NaOAc) to 28% buffer B over 60 minutes, followedby a second linear gradient to 100% buffer B over 20 minutes, andthen holding at 100% buffer B for a further 20 minutes.

Phospholipase C treatment and mild alkali hydrolysisDigestion with phosphatidylinositol-specific phospholipase C (fromBacillus thuringiensis) was performed in 20 mM Tris acetate, pH 7.5,containing 0.1% Triton X-100 at 37°C. For digestion with phospho-lipase A2 from bee venom, glycolipids were incubated in 0.1 M Tris-HCl, pH 7.4, containing 0.1% deoxycholate, 1 mM CaCl2 for 24 hoursat 37°C. Mild alkali hydrolysis was performed in 100 µl 0.1 Mmethanolic NaOH for 3 hours at 37°C. Samples were neutralized byadding 10 µl 1 M acetic acid, dried in a Speedvac evaporator, andanalyzed by HPTLC.

Chemical analysisNeutral sugars and lipids were measured by gas chromatography-massspectrometry (GC-MS) following methanolysis and trimethylsilyl(TMS) derivatization as described previously (McConville et al.,1990b). The myo-inositol content was measured by GC-MS after acidhydrolysis (6 N HCl, 110°C, 16 hours) and TMS derivatization.Selected ion monitoring for m/z 305 and 318 was used to quantitatethe myo-inositol content relative to a scyllo-inositol internal standard.Amino sugars and ethanolamine were determined using the WatersPico Tag system (Waters) as described by McConville et al. (1993).Methylation analysis of purified GIPLs was performed as describedby McConville et al. (1993).

The phosphate content of GIPLs was determined as described byBartlett (1959) with minor modifications: glycolipid mixtures weresubjected to HPTLC and the GIPLs located by staining withCoomassie Blue (Nakamura and Handa, 1984). GIPL-containingbands were scraped off the sheet and heated in 10 M H2SO4 for 3hours at 160°C. The samples were cooled to room temperature,supplied with H2O2 and again heated for 3 hours at 160°C. Thephosphate content was subsequently measured using phosphatidyl-choline as a standard.

Immunochemical techniquesNew Zealand white rabbits were immunized subcutaneously with1×109 live lesion-derived amastigotes in phosphate-buffered saline(PBS: 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.4 mMKH2PO4, pH 7.4). After 4 weeks booster injections followed at 2-3week intervals.

Antibodies directed against the surface of amastigotes were isolatedby incubating 2×109 cells in 50 ml Schneider’s Drosophila medium,pH 7.0, containing 20% iFCS with 1 ml antiserum for 1 hour at 4°C.Cells were recovered by centrifugation and washed once in cold

G. Winter and others

2473Glycolipids of

Leishmania mexicana

medium and twice in cold PBS. Cell surface bound antibodies wereeluted by incubating the cells in 10 ml 0.1 M glycine, 0.14 M NaCl,pH 2.75, for 15 minutes at 4°C. Cells were removed by centrifuga-tion and the supernatant immediately neutralized by adding 1 M Tris-Cl, pH 8.5. Thereafter, antibodies were concentrated and equilibratedto PBS by ultrafiltration and stored in a final volume of 1 ml at −20°C.The yield of antibodies was approximately 100 µg.

After HPTLC, immunostaining of glycolipids was performed asdescribed by Bethke et al. (1986). The sheets were immersed in 0.05%(w/v) polyisobutylmethacrylate in n-hexane for 1 minute, dried andthen incubated in blocking buffer (1% BSA, 0.05% Tween-20 in PBS)for 1 hour at 37°C. The sheets were overlayed with rabbit antibodiesdiluted in blocking buffer for 1 hour at 37°C, washed three times inPBS, 0.05% Tween-20 and then incubated with alkaline phosphatase-conjugated anti-rabbit IgG/IgM (1:5,000, Dianova, Hamburg, FRG)or anti-rat IgG/IgM (1:1,000, Dianova) in blocking buffer for 1 hourat 37°C. After three washings in PBS, 0.05% Tween-20, bound anti-bodies were visualized by overlaying plates with a 0.01% solution of5-bromo-4-chloro-3-indolylphosphate in 10 mM Tris, 0.14 M NaCl,2 mM MgCl2, 2 mM ZnCl2, pH 8.5.

Specific anti-glycolipid antibodies were isolated using glycolipidsseparated by HPTLC. HPTLC strips containing the desired glycolipidwere cut from the polyisobutylmethacrylate-coated sheets, incubatedwith blocking buffer and then with anti-amastigote serum diluted inblocking buffer for 1 hour at 37°C. After washing in PBS, 0.05%Tween-20 (3×) and PBS (1×), the silica gel was scraped off the sheet,suspended in PBS by sonication and loaded on a small column. Theantibodies were eluted from the gel with 0.1 M glycine, 1% BSA, pH2.75, at 4°C and immediately neutralized with 1 M Tris, pH 8.5.Elution of antibodies was assayed by enzyme-linked immunosorbentassay (ELISA) using chloroform/methanol/water extracts of amastig-otes as antigen. Antibody-containing fractions were pooled, equili-brated to PBS during ultrafiltration and stored at −20°C.

For ELISA analysis, chloroform/methanol/water extracts orHPTLC-purified glycolipids of 1×106 cells in 50 µl methanol wereapplied to each well of a 96-well polyvinylchloride microtiter plate(Titertek; Flow Laboratories, Meckenheim, Germany) and dried atroom temperature. Non-specific binding sites were blocked with PBScontaining 5% non-fat milk powder, 0.05% Tween-20. Plates werethen incubated with 100 µl/well alkaline phosphatase-conjugated goatanti-rabbit IgG/IgM antibodies in blocking buffer (1:5,000) for 1 hourat 37°C. After four washings, plates were developed with 1 Mdiethanolamine/1 mM MgCl2, pH 9.8, containing 1 mg/ml p-nitro-phenylphosphate. Phosphatase activity was determined spectrophoto-metrically at 405 nm. Gel electrophoresis and immunoblotting wereperformed as described by Ilg et al. (1993).

Immunofluorescence and immunoelectron microscopyImmunofluorescence of live or fixed parasites and infected peritonealmacrophages was performed as described previously (Stierhof et al.,1991). For cryostat sections, mouse lesions were fixed in 4%paraformaldehyde, 0.1% glutaraldehyde in 0.1 M PBS overnight at4°C. After washing the lesion tissue in PBS, single tissue pieces wereplaced into a drop of cryotomy embedding compound (OCT; Miles)on a cryostat stub and cooled to −20°C in a 2800 Frigocut E cryostat(Reichert-Jung). Sections (3-5 µm) were collected on 0.1% (w/v)poly-L-lysine (Sigma)-coated slides. Immunofluorescence on cryostatsections was performed as described for fixed parasites (Stierhof etal., 1991).

For immunoelectron microscopy mouse lesions were cut into smalltissue pieces (1-2 mm3) and fixed in 4% paraformaldehyde, 0.1% glu-taraldehyde in 0.1 M 1,4-piperazinediethanesulfonic acid (PIPES)buffer, pH 7.2, for 3 hours. Thereafter, the specimens were infiltratedwith 30% dimethylformamide in PIPES buffer (Meissner andSchwarz, 1990), plunged into liquid N2-cooled propane and trans-ferred into the freeze-substitution medium with 1% glutaraldehyde,0.5% osmium tetroxide, in anhydrous acetone. Freeze-substitution

was carried out in a FSU 030 freeze-substitution unit (Balzer) at−90°C for 48 hours, −60°C for 8 hours, and −40°C for 6 hours, wherethe specimens were washed three times in anhydrous acetone andinfiltrated with an acetone-Epon resin mixture. Subsequently,specimens were brought to room temperature, infiltrated with Eponresin, and polymerized at 70°C for 24 hours. Ultrathin sections weretreated with 50 mM glycine in PBS, blocked with 0.2% gelatin, 0.5%BSA in PBS, followed by incubation with antibodies and Protein A13 nm-gold (Slot and Geuze, 1985) for 1 hour at room temperature.After washing and contrasting with uranyl acetate and lead citratesections were examined in a Philips 201 electron microscope at 60kV.

RESULTS

Glycolipids are expressed at the surface ofamastigotesA rabbit antiserum raised against live, lesion-derived L.mexicana amastigotes intensely labeled the surface of thesecells (Fig. 1A; B, preimmune serum). No reaction was obtainedwith promastigotes (Fig. 1C) suggesting that amastigotesexpress antigens that are either not present in the insect stageor are shielded by surface components such as LPG. The rabbitserum was used to purify antibodies exclusively reactive withsurface components of amastigotes. Mouse-lesion-derivedparasites were incubated in culture medium containing anti-amastigote serum, and cell bound antibodies were eluted at pH2.75 in the cold. Remarkably, more than 90% of the amastig-otes survived this treatment as determined by Trypan Blueexclusion experiments and by their ability to differentiate topromastigotes in vitro. Furthermore, upon analysis by SDS-PAGE and staining with Coomassie Blue, the eluted proteinsshowed the typical pattern for immunoglobulins confirmingthat they contained insignificant amounts of amastigotepolypeptides. The purified antibody fraction strongly reactedwith the surface of live and fixed amastigotes (data not shown).

In immunoblots, the rabbit antiserum recognized a broadrange of proteins from both developmental stages (Fig. 2A).The purified anti-amastigote-surface antibodies exclusivelylabeled antigens near the front of a blot of amastigote lysates(Fig. 2B, lane 1), whereas a corresponding band was not oronly weakly detectable in a blot of promastigote lysates (lane2). Preimmune serum did not react with any parasite antigen(Fig. 2C). Treatment of amastigotes with chloroform/methanol/water and analysis of the extract and the insolubleresidue by immunoblotting showed that the antigens movingto the front of the gel were exclusively associated with theorganic solvent fraction (results not shown). This suggestedthat antibodies against the surface of amastigotes reacted withlow molecular mass and lipophilic compounds. Candidateantigens were glycolipids. Glycolipids present in the chloro-form/methanol/water extract of promastigotes or amastigoteswere resolved by HPTLC and stained by orcinol-H2SO4.Adopting the nomenclature of McConville et al. (1993), thepromastigote extract contained the previously identified GIPLsiM2, iM3, iM4 and EPiM3 (for structures see Table 1); theamastigote extract showed four corresponding glycolipids,which had slightly higher Rf-values (Fig. 3A, lanes 2 and 1,respectively). In addition, amastigote extracts gave rise to adouble band at an Rf-value of about 0.6; it will be shown below

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that this double band corresponds to the Forssman glycosph-ingolipid (GSL). Immunostaining of HPTLC plates demon-strated which glycolipids were recognized by the antibodies(Fig. 3B). In the case of amastigotes, EPiM3 and ForssmanGSL gave rise to a reaction with both the anti-amastigoteserum (lane 1) and derived anti-surface antibodies (lane 2); aminor component designated GSL-X was not analysed further.In contrast, EPiM3 was the only component reactive in the pro-mastigote extract (lane 3). Preimmune serum did not react(lane 4).

Structural analysis of amastigote glycoinositolphospholipidsThe structures of the amastigote GIPLs were analyzed by themethodology described by McConville et al. (1993). Firstly,

treatment of the glycolipid fraction with phosphatidylinositol-specific phospholipase C (Fig. 4A,B, lanes 2) degraded thecomponents provisionally designated iM2, iM3, iM4 andEPiM3. Secondly, after mild alkali (Fig. 4A, lane 3) or phos-pholipase A2 treatment (data not shown) we obtained a ladderof bands with reduced Rf-values that comigrated with the dea-cylated forms of promastigote iM2, iM3, iM4 and EPiM3 (datanot shown). These results suggested that the four amastigoteGIPLs contain a phosphatidylinositol lipid moiety with 1-alkyl-2 acylglycerol. Interestingly, the reaction of EPiM3 withanti-amastigote surface antibodies was abolished after mildalkali treatment (Fig. 4B, lane 3, see also McConville et al.,1990a). In a third step, GIPL glycans released by phospholi-pase C treatment were analysed by Dionex HPLC after nitrousacid deamination and radioactive labeling by NaB3H4reduction. This procedure generated four major labeled glycansfrom the amastigote fraction that had the same relativeretention times as the glycans of promastigote iM2, iM3, iM4and EPiM3, but in markedly different ratios (Fig. 5). The originof each of the labeled glycans was confirmed by nitrous aciddeamination and NaB3H4 reduction of HPTLC-purified GIPLs.The HPLC profile of the released glycans also indicated thatthe amastigotes lack detectable levels of type-2 GIPLs. Theglycans of these GIPLs eluted as a minor complex at ~6 DUin Dionex HPLC of promastigote GIPL glycans. Fourthly,three of the amastigote GIPLs (EPiM3, iM3, iM4) werepurified by HPTLC and subjected to compositional analysis,methylation linkage analysis and exoglycosidase sequencing(results not shown). These experiments showed that theirglycan structure was identical to the corresponding promastig-ote GIPLs (Table 1). Finally, lipid analysis demonstrated thatthe 1-O-alkylglycerol moieties of the amastigote GIPLscontained predominantly C18:0 alkyl chains (98-99%). Thepromastigote GIPLs have the same alkyl chain composition(McConville et al., 1993), suggesting that the slight differencesin the HPTLC Rf values of the promastigote and amastigoteGIPLs (Fig. 3A) reflect differences in the acyl-chain composi-tion.

As deduced from quantitation of the Dionex-HPLC profile(Fig. 5), the relative abundance of the four GIPLs was quite


Fig. 1. Reactivity of anti-amastigote rabbit serum with fixedamastigotes and promastigotes. Lesion-derived amastigotes (A,B) orpromastigotes (C) were treated with anti-amastigote (A,C) orpreimmune serum (B) and fluoresceine-isothiocyanate (FITC)-labeled secondary antibodies. The left panels show theimmunofluorescence, the right panels the DNA staining with 4,6-diamidino-2-phenylindole (DAPI). Bar, 20 µm.

Fig. 2. Reactivity of anti-amastigote serum and anti-amastigote-surface antibodies in immunoblots. Lysates (50 µg protein) fromamastigotes (lanes 1) and promastigotes (lanes 2) were separated on7.5-20% SDS-PAGE gels, blotted to polyvinylidenedifluoridemembranes and probed with anti-amastigote serum (A), anti-amastigote-surface antibodies (B) or preimmune serum (C). Standardproteins in kDa are indicated at the right.

2475Glycolipids of Leishmania mexicana

different in promastigotes and amastigotes (Table 1, comparealso orcinol-H2SO4 staining pattern in Fig. 3A). The predom-inant component in amastigotes was EPiM3, which, based onphosphate determinations, amounted to 1.8×107 molecules/cell. This glycolipid was only a minor component in themixture of promastigote GIPLs (Table 1, McConville et al.,1993).

Lesion-derived amastigotes contain host-derivedForssman glycosphingolipidThe glycolipid provisionally designated Forssman glycosphin-golipid in Fig. 3 was resistant to the treatment with phospho-lipase C, mild alkali (Fig. 4) or phospholipase A2. These prop-erties are typical for GSLs. McConville and Blackwell (1991)showed that L. donovani amastigotes isolated from spleens of

Syrian hamsters contain a host-derived GSL homologous to thepara-Forssman GSL of human erythrocytes (GalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc1-Ceramide). Evidence fora similar GSL in the mouse amastigote preparation wasinitially obtained by fast atom bombardment mass spectrome-try of the peracetylated glycolipid, which showed a signal char-acteristic for the terminal oligosaccharide sequence HexNAc-HexNAc (J. Peter-Katalinic and G. Winter, unpublishedresults) present in the Forssman GSL (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc1-Ceramide; Siddiqui andHakomori, 1971). Therefore, we tested whether the rat mAbIII E2 against Forssmann GSL from sheep erythrocytesdescribed by Bethke et al. (1987) would react with the GSL ofthe L. mexicana amastigote extract. Globoside (GalNAcβ1-3Galα1-4Galβ1-4Glc1-Cer), Forssman GSL and amastigote

Table 1. Structure and abundance of GIPLs in L. mexicana amastigotes and promastigotesGIPL Structure Amastigotes Promastigotes

iM2 Manα1-3Manα1-4GlcNα1-6Pl 10 27Manα1-6

iM3 Manα1-4GlcNα1-6Pl 13 13Manα1-3

Manα1-2αManα1-6iM4 Manα1-4GlcNα1-6Pl 11 48

Manα1-3

EtNPO4-Manα1-6EPiM3* Manα1-4GlcNα1-6Pl 63 9

Manα1-3

Relative amounts were determined from the radioactivity profiles shown in Fig. 5, which correspond to the GIPLs after phase partitioning into amethanol/water phase (see Materials and Methods). The values obtained with promastigotes are in agreement with previous results (McConville et al., 1993).

*For location of ethanolamine phosphate see ‘note added in proof’.

Fig. 3. Analysis of glycolipids by HPTLC and immunostaining.(A) Glycolipid extracts of 2×108 amastigotes (lane 1) orpromastigotes (lane 2) were stained with orcinol-H2SO4. In additionto the labeled bands variable amounts of minor components weredetected in amastigote extracts, which are likely due to contaminatinghost glycolipids. The stain at the origin (bottom of HPTLC) wascaused by glycosylated non-lipid material present in thechloroform/methanol/water-extracts. Glycolipids of amastigotes orpromastigotes are indicated by A- and P-, respectively.(B) Amastigote extracts were probed with anti-amastigote serum (lane1) or anti-amastigote-surface antibodies (lane 2). Lane 3 refers to apromastigote extract probed with anti-amastigote-surface antibodies,lane 4 to an amastigote extract probed with preimmune serum.

Fig. 4. Susceptibility of amastigote glycolipids tophosphatidylinositol-specific phospholipase C and mild alkalitreatment. Glycolipid mixtures before (lanes 1) and afterphospholipase C (lanes 2) or mild alkali treatment (lanes 3) wereseparated by HPTLC and stained with orcinol-H2SO4 (A) orincubated with anti-amastigote-surface antibodies (B).

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glycolipids were separated by HPTLC and either stained withorcinol-H2SO4 (Fig. 6A) or incubated with mAb III E2 (B) oranti-amastigote-surface antibodies (C). The amastigote GSLand authentic Forssman GSL showed the same Rf values anda mutual reaction with the antibodies while globoside, thebiosynthetic precursor of the Forssman GSL, was not recog-nized. Furthermore, mAb III E2 does not react with para-Fossman GSL (P. Mühlradt, personal communication). Theseresults strongly suggested that the L. mexicana amastigotepreparation contains Forssman GSL.

In order to investigate whether the parasites themselves syn-thesize Forssman GSL or whether this lipid originates from thehost, amastigotes from mouse lesions were grown axenicallyat 34°C and lipid extracts from cells taken at various timeswere analyzed by HPTLC and immunostaining with anti-amastigote-surface antibodies (Fig. 7A). The Forssman GSLand the undefined GSL-X (compare also Fig. 3) disappearedwithin 72 hours of cultivation suggesting that these GSLs werenot synthesized by the parasites. In contrast, the level of EPiM3remained the same showing that the rate of synthesis of thisglycolipid was not altered significantly.

Cellular localization of EPiM3 and Forssmann-GSLon lesion-derived and cultured amastigotesThe detection of Forssman GSL in the amastigote preparation

raised the question whether this lipid indicated the presence ofcontaminating membranes from host cells or whether it was acomponent of the parasite membrane. Therefore, antibodiesagainst Forssman GSL and EPiM3 were affinity-purified fromthe anti-amastigote serum. When tested by ELISA, no cross-reaction was observed. As shown by immunofluorescenceexperiments on live (data not shown) and fixed lesion-derivedamastigotes, the purified anti-Forssman GSL antibodiesreacted with the entire surface of all parasites (Fig. 7B) andthis reaction disappeared gradually in cultured amastigoteswithin 72 hours (Fig. 7C,D). Purified anti-EPiM3 antibodiesalso reacted with the cell surface but expression of this antigenwas not altered during cultivation (Fig. 7E,F). As a measurefor host cell contaminations, Fig. 7G and H show the reactionwith mAb IG11, which is directed against the LAMP1 protein,a major component of the lysosomal membrane ofmacrophages (Rabinowitz et al., 1992). This antibody did notrecognize the majority of freshly isolated amastigotes.However, some cells showed fluorescence frequently in theform of a cap attached to the posterior end of the parasites (Fig.7G). However, the reaction with this antibody was lost afterculturing the cells for only 24 hours (Fig. 7H).

Taken together, the experiments described in Fig. 7 allowedthe following conclusions. Firstly, EPiM3 is a parasitecomponent located at the cell surface. Secondly, ForssmanGSL appears not to be synthesized by amastigotes but likelyis inserted into the parasite cell membrane. Thirdly, purifiedlesion-derived amastigotes are contaminated by fragments ofmacrophage lysosomal membranes. Since L. mexicanaamastigotes are frequently attached to the membrane of theparasitophorous vacuole (Antoine et al., 1990; Stierhof et al.,1991; see also Fig. 9), membrane fragments may either remainassociated with the amastigotes during tissue homogenizationor they may bind thereafter.

Cellular localization of EPiM3 and Forssman GSL ininfected macrophages and mouse lesionsThe assumed transfer of Forssman GSL from the macrophage


Fig. 5. HPLC analysis of the GIPL glycans of amastigotes andpromastigotes. Glycolipids of amastigotes (A) and promastigotes (B)were extracted with chloroform/methanol/water, partitioned into amethanol/water phase and purified by octyl-Sepharosechromatography. After treatment with phospholipase C the releasedglycans were radiolabeled by nitrous acid deamination/NaB3H4-reduction prior to analysis by Dionex HPLC. The elution positions ofthe labeled glycans were determined relative to a series of co-injected dextran oligomers (expressed as Dionex Units, DU). Peakseluting beyond 8.6 DU represented complex deamination products ofthe ethanolamine-containing EPiM3 molecule and were alsoobtained with HPTLC purified amastigote and promastigote EPiM3.

Fig. 6. Occurrence of Forssman GSL in lesion-derived amastigotes.Globoside from human erythrocytes (5 µg; lanes 1), Forssman GSLfrom sheep erythrocytes (5 µg; lanes 2) and the organic solventextract corresponding to 2×107 amastigotes (lanes 3) were subjectedto HPTLC and stained with orcinol-H2SO4 (A), incubated with theForssman GSL-specific mAb III E2 (B) or the anti-amastigote-surface antibodies (C).


to the amastigote surface could be an artifact of the isolationprocedure. Therefore, we performed experiments to localizethe glycosphingolipid and EPiM3 in infected macrophages andin lesion material. In peritoneal macrophages infected 10 days

earlier with cultured amastigotes, the affinity-purified anti-EPiM3 antibodies bound to the surface of the parasites (Fig.8A,B), while the affinity-purified anti-Forssman GSL anti-bodies gave no specific reaction (Fig. 8C,D). Cryostat sections

Fig. 7. Loss of Forssman GSL and host cell contaminants during cultivation of amastigotes in vitro. Amastigotes isolated from mice werecultivated at 34°C in vitro. At various times, 5×107 cells were extracted with chloroform/methanol/water and subjected to HPTLC and stainingwith anti-amastigote-surface antibodies (part A: lane 1, 0 hours; lane 2, 24 hours; lane 3, 48 hours; lane 4, 72 hours). Alternatively, sampleswere processed for immunofluorescence using affinity-purified anti-Forssman GSL antibodies (B, 0 hours; C, 24 hours; D, 72 hours), affinity-purified anti-EPiM3 antibodies (E, 0 hours; F, 72 hours) or the anti-LAMP1 mAb IG11 (G, 0 hours; H, 24 hours). Bar, 10 µm.

Fig. 8. Absence of Forssman GSL in amastigotes of infected peritoneal macrophages. Forssman GSL-negative culture amastigotes were used toinfect peritoneal macrophages. After 10 days, the cells were fixed, permeabilized and subjected to indirect immunofluorescence using anti-EPiM3 antibodies (A); the same cell stained for DNA with DAPI (B); or anti-Forssman GSL antibodies (C; DAPI staining in D). Arrows in Band D point to the comma-shaped kinetoplast of amastigotes. n, nucleus; pv, parasitophorous vacuole. Bar, 20 µm.

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of mouse lesions provide an overview of many heavily infectedcells (Fig. 9). After double-labeling with anti-EPiM3 anti-bodies and mAb IG11, the former antibodies recognized thesurface of amastigotes but not the host tissue, while the mAbreacted with host cell membranes (Fig. 9A). The para-sitophorous vacuole membrane appeared to be tightly attachedto part of the amastigotes. The affinity-purified anti-Forssmanantibodies (Fig. 9B) reacted with both the host tissue and thesurface of the amastigotes, which were not labeled by the mAbIG11 (see legend of Fig. 9 for a detailed interpretation). Theseresults were confirmed at a higher resolution by immunoelec-tron microscopy. The anti-EPiM3 antibodies were localized atthe amastigote surface and on membranes lining the flagellarpocket and the extended lysosomes but not on host cell struc-tures (Fig. 10A) while the rabbit anti-Forssman antibodieslabeled the same amastigote structures and host cellmembranes (Fig. 10B). The mAb III E2 revealed a similar butweaker labeling pattern of the Forssman antigen (Fig. 10C). Ingeneral, the labeling density of amastigotes and host cellsvaried, suggesting different levels of antigen expression. Cellsother than macrophages were not labeled with the mAb III E2(Fig. 10C).

DISCUSSION

GIPL structure and biosynthesisThe present study shows that the GIPL profiles of L. mexicanapromastigotes and amastigotes are qualitatively similar butquantitatively distinct. While the major GIPLs of promastig-otes have neutral glycan headgroups, the predominant GIPL ofthe amastigotes, EPiM3, is substituted with the zwitterionicethanolamine-phosphate group. The amastigote GIPL profile

also differs from the promastigote profile in lacking detectablelevels of the type-2 GIPLs. At least some of these GIPLs arelikely to be precursors to promastigote LPG (McConville et al.,1993). The absence of these GIPLs in the amastigotes, whichalso lack detectable levels of LPG (Bahr et al., 1993), providesfurther support for their precursor role and suggests that thedownregulation of LPG coincides with the repression of oneor more enzymes involved in the synthesis of the type-2 GIPLsfrom the pool of iM2. A candidate regulatory enzyme wouldbe the putative Galfβ1-3-transferase that forms GIPL-1(Galfβ1-3Manα1-3Manα1-4GlcNH2α1-6PI) from iM2. Asimilar situation may occur in L. donovani amastigotes.However, in this species the amastigote GIPLs lack the 3-linked mannose branch (McConville and Blackwell, 1991),suggesting either that synthesis of the LPG core is controlledat an earlier stage, by the Manα1-3-specific transferase, or thatthe L. donovani amastigote GIPLs are additionally processedby an α1-3-specific mannosidase. L. major amastigotes differfrom both L. mexicana and L. donovani amastigotes in contin-uing to synthesize type-2 GIPLs and also low levels of LPG(Schneider et al., 1993; Moody et al., 1993) further supportingthe notion of a precursor-product relationship.

GIPLs and the structure of the amastigote surfaceOn the ultrastructural level, the amastigote surface appears asa plain unit membrane without discernable coat (Fig. 10,compare also Pimenta et al., 1991 and Stierhof et al., 1991).Its biochemical characterization is in an unsatisfactory state inspite of the fact that many groups have tried to characterizesurface components by either labeling techniques or by use ofmonoclonal antibodies (Handman and Hocking, 1982;Handman and Curtis, 1982; Sadick and Raff, 1985; Pan andMcMahon-Pratt, 1988; Jaffe and Rachamim, 1989; Medina-


Fig. 9. Occurrence of Forssman GSL in lesionamastigotes. Immunofluorescence on cryostatsections of mouse lesions. (A and B) Double-labeling experiments with anti-EPiM3antibodies (A; Cy3-conjugated secondaryantibodies, red fluorescence) or anti-Forssmanantibodies (B; red fluorescence) and the anti-LAMP1 mAb IG11 (FITC-conjugatedsecondary antibodies, green fluorescence).Superposition of the two fluorophoresemissions results in a yellow fluorescence. (A)The arrowheads point to amastigotes in theparasitophorous vacuole (pv); the parasitesurface contains EPiM3 but not LAMP1 (redfluorescence). The large arrows point to hostmembranes containing LAMP1 (greenfluorescence); regions in the attachment zonebetween amastigotes and the pv membrane(small arrows) contain both antigens (yellowfluorescence). (B) Amastigotes in the pv show astrong labeling with anti-Forssman antibodies(arrowheads); the antigen is also present in thehost tissue (large arrows). The attachment zonegives a yellow fluorescence (small arrows)indicating the presence of both Forssman GSLand LAMP1. Omission of the primaryantibodies did not give a reaction. Bar, 10 µm.


Acosta et al., 1989; Eperon and McMahon-Pratt, 1989). Amajor difficulty lies in the choice of cell preparation used forthe investigations. When working with lesion-derived amastig-otes one has to rigorously exclude contamination by host cellmembranes. As demonstrated here (Fig. 7), the antibody

against the major protein, LAMP1, of macrophage lysosomalmembranes provides a sensitive reagent for detecting particu-late impurities. In addition, the parasite may contain solubleproteins such as antibodies adsorbed to the surface. The alter-native is to use amastigotes cultured in vitro (see Bates, 1993,

Fig. 10. On-section immunogold-labeled ultrathin resin sections oflesions from L. mexicana-infectedBalb/c mice. (A) Labeling with affinity-purified anti-EPiM3 antibodies. Thelabel is found on the cell surface ofamastigotes residing in theparasitophorous vacuole (pv), on themembrane of the extended lysosomes (l)and in the flagellar pocket (not shown),but not on macrophages (m). (B)Labeling with affinity-purified anti-Forssman GSL antibodies revealedstrong labeling of the infectedmacrophage. In addition, the antigen islocated on the surface, in the flagellarpocket (not shown), and the lysosomalmembranes of amastigotes. (C) ThemAb III E2 recognizes the Forssmanantigen in infected macrophages as wellas the amastigote surface, lysosomesand the flagellar pocket membranes (fp).No specific labeling was obtained whenthe antibodies were omitted. fl,flagellum; n, nucleus. Bar, 1 µm.

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for review). Although such parasites retain the morphology oflesion-derived amastigotes, their biosynthetic machinery maychange in the direction typical for promastigotes. Thus, wehave observed repeatedly that cultured L. mexicana amastig-otes from mouse lesions synthesize considerable amounts ofLPG, which is not formed as long as the parasites reside in themammal (T. Ilg, unpublished data). Therefore, results obtainedwith cultured amastigotes must be interpreted cautiously.

The present study defines the glycolipid EPiM3 as a surfacecomponent of L. mexicana amastigotes. The parasites containabout 3×107 molecules of GIPLs/cell, of which approximately60% account for EPiM3. The percentage of GIPLs present atthe L. mexicana amastigote surface remains unknown.However, taking an amastigote surface area of 3×107 nm2 andan area of 3 nm2 for the glycan head group of the GIPLmolecules, it is clear that 1/3 of the cell-associated GIPLs couldcover the entire cell surface (McConville and Blackwell,1991), the rest being located in intracellular membranes suchas lysosomes. The GIPL glycans at the surface of amastigotesmay form a dense hydrogen-bonded network, which couldserve the required protective function against hydrolasessimilar to the GSLs present on the apical face of epithelial cellsin the digestive tract of mammals (Simons and van Meer,1988). EPiM3 is not transferred from the parasite surface tothe host cell (Fig. 10A). Interestingly, glycolipids related to theGIPLs are also found in high amounts at the surface of otherkinetoplastidae living in a hydrolytic environment (seeMcConville and Ferguson, 1993, for review). These glyco-lipids contain either ethanolamine-phosphate or a relatedresidue, 2-aminoethylphosphonate, linked to the core glu-cosamine. However, a similar modification was not observedin L. donovani amastigote GIPLs (McConville and Blackwell,1991). Therefore, the functional significance of this substitu-tion is unclear.

Immunoblotting using rabbit anti-amastigote-surface anti-bodies have so far not led to the detection of candidate surfaceproteins (Fig. 2). A trivial reason for this failure could be thatthis polyvalent serum contained only antibodies against con-formational epitopes, which are destroyed during samplepreparation for SDS-PAGE. Attempts to immunoprecipitateputative surface proteins from a membrane extract of meta-bolically labeled cells prepared in 0.5% sodium dodecyl sulfateand diluted with 0.5% Nonidet P40 before treatment with anti-amastigote-surface antibodies were unsuccessful (Braunger,1993). Therefore, we favor the possibility that amastigotescontain no predominant surface protein that is accessible toantibodies. In fact, a major surface protein serving a protectivefunction may not be present at all considering that labelingtechniques applied by several groups (Medina-Acosta et al.,1989; Sadick and Raff, 1985) including our own (unpublishedresults) have failed to provide any evidence for its existence.The plasma membrane of amastigotes may therefore be uniquein having mainly multi-membrane-spanning proteins burried inthe lipid bilayer, which are required, for example, for nutrientuptake (Glaser and Mukkada, 1992). Such proteins aregenerally poorly immunogenic and difficult to detect bysurface labeling reagents.

Forssman GSL synthesis and transfer toamastigotesThe lesions induced in mice contain high amounts of Forssman

GSL both in the host tissue and on the surface and on internalmembranes of the amastigotes. It has been proposed thatamastigotes themselves have the ability to form glycosphin-golipids (Barbieri et al., 1993; Strauss et al., 1993).McConville and Blackwell (1991) detected hamster-derivedpara-Forssman GSL in L. donovani amastigote preparations,but could not metabolically label this lipid with [3H]glucose.In accordance with these authors, we consider the ForssmanGSL associated with L. mexicana amastigotes to be synthe-sized by the host rather than the parasite. This is suggested bythe selective loss of the GSLs, but not the GIPLs from lesion-derived amastigotes cultured in vitro. In addition, these GSL-negative amastigotes do not acquire Forssman antigen afterinfection of macrophages in vitro.

In healthy mice, Forssman GSL is present on macrophagesin the spleen and peripheral lymph nodes but not in residentperitoneal macrophages (Bethke et al., 1987, compare Fig.8C,D). Interestingly, Forssman GSL can be induced in vitro byincubation of peritoneal macrophages with interleukin 4 (IL4,Kleist et al., 1990). Furthermore, the uncontrolled infection ofBalb/c mice with L. major leads to the expansion of IL4-secreting TH2 cells (Reed and Scott, 1993). Since the infectionof these mice with L. mexicana is also uncontrolled, it ispossible that they as well form T cell-derived IL4, which inturn induces the synthesis of Forssman GSL.

The uniform distribution of the Forssman antigen over thesurface of purified amastigotes in contrast to the patchy dis-tribution of the LAMP1 glycoprotein, which acts as a markerof contaminating parasitophorous vacuole membranessuggests a remarkable transfer of GSL from the host cell tothe parasite. This notion is supported by immunofluorescenceand immunoelectron microscopic studies of sectioned mouselesions. The transfer may occur by some kind of flip-flop inthe attachment zone of the amastigotes with the phagolysoso-mal membrane (Antoine et al., 1990; Stierhof et al., 1991;Lang et al., 1994) or by monomeric, micellar or vesiculartransport through the lumen of the vacuole. The mechanismof transfer of the GSL, its mode of association with theparasite surface as well as the functional consequences of thisphenomenon for the spread of the infection will require furtherexperimentation.

We thank Gareth Griffiths for the anti-LAMP1 antibody, PeterMühlradt for the anti-Forssman GSL antibody, Jana Peters-Katalinicand Heinz Egge for mass-spectrometric measurements, Thomas Ilgfor helpful comments on the manuscript, Gerda Müller and AndreaJacob for the photography and the Deutsche Forschungsgemeinschaftfor support. M.J.M. is a Wellcome Trust Senior Research Fellow.

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(Received 7 March 1994 - Accepted 4 May 1994)

Note added in proofFurther analysis of the promastigote and amastigote GIPLs byfast atom bombardment-mass spectrometry confirmed thepresence of stage-specific differences in the fatty acid compo-sition of the alkylglycerol moieties (McConville and Currie,unpublished data). While the major fatty acid in the pro-mastigote GIPLs was C14:0, the major fatty acid in theamastigote GIPLs was 16:0. In contrast, the alkyl chain com-position (predominantly C18:0) was the same in both the pro-mastigote and amastigote GIPLs. These analyses also provided

information on the location of the phosphoethanolamineresidue in the amastigote EPiM3. In addition to the molecularion of the major molecular species, EtNH2PO4Hex3HexNH2PI(m/z 1593), fragment ions at m/z 1308 and 984 were observedthat correspond to Hex2HexNH2PI and HexNH2PI, respec-tively. This fragmentation pattern suggests that theethanolamine phosphate residue is located on one of theterminal mannose residues of iM3, and not on the GlcNresidue, as originally proposed for promastigote EPiM3(McConville et al., 1993). Moreover, treatment of the radiola-beled glycan moiety (prepared by nitrous acid deamination andNaB3H4 reduction) with Jack bean α-mannosidase beforehydrofluoric acid dephosphorylation, removed the Manα1-3branch but not the Manα1-6 branch, indicating that theethanolamine phosphate was located predominantly or exclu-sively on the Manα1-6 branch.


surface antigens of leishmania mexicana amastigotes

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