THEJOURNALOF BIOLLXICALC~ISTRY Vol. 269, No. 4, Issue of January 28, pp.

2597-2606, 1994
0 1994 by The American Soeiety for Biochemistry and Molecular Biology, Inc Printed in U.S.A.

Glycosylphosphatidylinositols Synthesized by Asexual Erythrocytic

Stages of the Malarial Parasite,Plasmodium falciparum
CANDIDATES FOR PLASMODIAL GLYCOSYLPHOSPHATIDYLINOSITOL MEMBRANE ANCHOR PRECURSORS
AND PATHOGENICITY FACTORS*

(Received forpublication, March 31, 1993, and in revised form, September 6, 1993)

Peter GeroldS, Angela Dieckmann-Schuppert, and Ralph T. Schwarz

From Zentrum fur Hygiene und Medizinische Mikrobiologie, Philipps- Universitat Marburg, Robert-Koch-Strasse 17,
35037 Marburg, Germany

Plasmodium falciparum is the causative agent of ma- the plasma membrane, in a wide range of organisms. A com-
laria tropica in man. Biochemical studies were focused parison of the glycan structures of GPI membrane anchors
on the asexual, intraerythrocytic stages of E? falcipa- indicates that the glycan parts of GPI-anchors contain a con-
rum, because of their role in the clinical phase of the servedstructure consisting of ethanolamine-P04-6Manal-
disease and the possibility of propagation in a cell cul-2Manal-GManal-4GlcN (for review see Cross (1990)).
ture system. In this report, we describe the in-culture A huge variety of modifications on the periphery of the core
labeling of malarial glycolipids and the analysis of their glycan structure havebeen observed. However, the core glycan
hydrophilicmoieties.Theywereidentified as glyco- of protein-linked GPIs is highlyevolutionarily conserved
sylphosphatidylinositols (GPIs)by: l) labelingwith throughout mammalian and protozoan cells. Therefore, it is
[SH]mannose,[SH]glucosamine,and[SHlethanolamine likely that identical or similarbiosynthetic pathways are used
and 2) sensitivity toward glycosylphosphatidylinositol- in all eukaryotes.
specific phospholipaseD, phospholipaseA2, and nitrous The biosynthesis of the trypanosoma1 GPI has been eluci-
acid. Malarial GPIs are shown to be unaffected by treat- dated usinga cell-free system from lkypanosoma brucei brucei
ment with phosphatidylinositol-specificphospholipase (Masterson et al., 1989; Doering et al., 1990; Menon et al.,
C, regardless of prior treatment with mild base com- 1990a) and was also elucidated for some other biological sys-
monly used for inositol deacylation. Two candidates for tems (Puotiet al.,1991; Stevens and Raetz, 1991; Tomavo et al.,
putative GPI-anchor precursors to malarial membrane 1992). GPI synthesis starts with the transferof GlcNAc from
proteins with the structures ethanolamine-phosphate- UDP-GlcNAc to PI, followed by de-N-acetylation. Three man-
6(Manal-2)Manal-2Manal-6Mana14GlcN-P1 (Pfgl a) nose residues, each derived from dolichol-phosphate mannose,
and ethanolamine-phosphate-6Manal-2Manal-6Man-are then added,yielding MansGlcN-PI. Phosphoethanolamine,
al-4-GlcN-PI (Pfgl /3) were identified. derived fromphosphatidylethanolamine (Menon et al., 1993), is
added to the terminal mannose to complete the GPI-anchor
precursor which is thought to be added to the protein in ex-
Malaria is the disease caused by parasitic protozoa of the change for a C-terminal hydrophobic domain in a transamidase
genus Plasmodium. Plasmodium falciparum,the most danger- reaction (Cross, 1990).
ous of the four speciesthat are naturallyinfectious to man, still Like many parasitic protozoa, I! falciparum has a complex
remains the cause of one of the most important diseases in life cycle involving an insect vector (Anopheles spp. ). During
tropical and subtropical areas. In1985, the WHO reported 4.8 the development in man, the intraerythrocytic, asexual stages
million documented cases of malaria, andmore than 50% of the produce the merozoite surface protein 1(MSPl), themerozoite
world’s population live inareaswheremalaria is endemic surface protein 2 (MSPO), and other proteins (Holder et al.,
(Lockyer and Holder, 1989). 1985; Haldar et al., 1985; Haldar et al., 1986; Smythe et al.,
Glycosylphosphatidylinositols (GPIs)’ are a recently discov- 1988; Braun-Breton et al., 1988), which were shown to be mem-
ered class of glycolipids that anchor proteins and sugars into brane-anchored involving diacylglycerol. These data lead to the
proposition that some I! falciparum surface proteins are an-
*This study was supported by DeutscheForschungsgemeinschaft chored by a glycosylphosphatidylinositol (GPI) membrane an-
Grants Schw 296/4-1, Schw 296/4-2, and Schw 296/4-3, Fondsder
Chemischen Industrie, ARC from British CounciVDAAD,Hessisches chor to the parasite membrane (Haldaret al., 1985; Haldar et
Ministerium fiir Wissenschaft und Kunst,and P. E. Kempkes Founda- al., 1986; Schwarz et al., 1986; Schwarz et al., 1987).
tion Marburg, Germany.This communication is presented as a partial It hasrecently been demonstrated that the GPI moiety may
fulfillment of the requirements of the doctoral thesis of P. G. The costs also play an importantrole in thepathology of severe malaria,
of publication of this article were defrayed in part by the payment of in addition to its role in membrane anchoring of surface pro-
page charges. This article must therefore be hereby marked “aduertise-
ment” in accordance with 18 U.S.C. Section 1734 solely toindicate this teins. Purified C-terminal GPI derived from MSPl and MSP2
fact. by pronase hydrolysis induces tumor necrosis factor and inter-
This work is dedicated to the memory of Bernard Fournet (1940- leukin 1production by macrophages and regulatesmetabolism
1993) to whom the field of glycoconjugates owes so much. in adipocytes. When administered to mice in uiuo, it induces
$ Recipient of a scholarship of the Hessische Graduiertedorderung.
To whom correspondence and reprint requests should be addressed: cytokine release, transient pyrexia, and hypoglycemia and un-
Tel.: +49-6421-285149or +49-6421-285493;Fax: +49-6421-285482. der some conditions elicits cachexia (Schofield and Hackett,
Theabbreviations used are: GPI,glycosylphosphatidylinositol; 1993).
MSP, major surface protein; PI-PLC, phosphatidylinositol-specificphos- In this report we provide evidence that the biosynthesis of
pholipase C; PIA,, phospholipase Az; GPI-PLD, glycosylphosphatidyl-
inositol-specificphospholipase D; HPAEC,highpHanionexchange GPIs protozoan I! falciparum involves a hydro-
in the parasitic
chromatography; Dol(?)-P-Man, dolichol(?)-phosphate-mannose (see phobically modified inositol ring and therefore shows a simi-
Footnote 2); Pfg,, Z? fakiparum glycolipid; AHM, 2,5-anhydromannitol. larity to the pathways described for mammalian cells (for a
2597

This is an Open Access article under the CC BY license.

2598 Glycosylphosphatidylinositols
Plasmodium
falciparum
in
review see Englund (1993)).The malarial pathway leads to two methanoUO.l% KC1 (10:10:3, by volume).
putative GPI-anchor precursors (ethanolamine-phosphate- Analysis and Purification of Glycolipids-The Folch-washed CM ex-
6Manal-2Mana1-6Mana14GlcN-PIand ethanolamine-phos- tracts andthe butanol components wereanalyzed on Silica Gel60 TLC
phate-6(al-2Man)Manal-2Mana1-6Mana14GlcN-PI)whose plates (Merck) using solvent systems A and B, respectively. After chro-
matography, the plates were dried and scanned for radioactivity using
glycan structures differ only in the number (3 versus 4) of
a Berthold LB 2842automatic "LC scanner. The areas corresponding to
mannose residues. the peaks of radioactivity were scraped, and the glycolipids were ex-
EXPERIMENTALPROCEDURES tracted twice with chlorofodmethanol (2:1, v:v),
twice with
chlorofodmethanollwater (10:10:3,v:v:v), and once with methanov
Materials-~-[6-~H]Glucosamine hydrochloride (26.0 Ci/mmol), ~ 4 2 - pyridinelwater (2:1:1,v:v:v) for 30 min each under sonification. The
3Hlmannose (13.6 Ci/mmol),[9,10-3H]myristicacid (50.0Ci/mmol), yield of purified glycolipids was estimated to be approximately 40%.
[9,10-3Hlpalmiticacid(53.4Ci/mmol), my~-[~H]inositol (108.4 Ci/ Acid Hydrolysis for Component Analysis-TLC-purified glycolipids
mmol), and [l-3Hlethan-l-ol-2-amine hydrochloride(29.5Ci/mmol) were dried to the bottom of a Reacti-vial (Pierce) and hydrolyzed for4 h
were purchased fromAmersham.Tunicamycin was purchased from in 4 N HCl at 100 "C. HCl was removed from the samples by repeated
Calbiochem. Phosphatidylinositol-specificphospholipase C (PI-PLC)
flash evaporation with 100 pl of methanol. The released monosaccha-
from Bacillus cereus and phospholipase A, (PLA,) from bee venom were
rides were analyzed by Dionex-HPAEC (Dionex, Sunnyvale, CA) using
fromBoehringer Mannheim. Jack bean a-mannosidase was from
elution program 2 (see below).
Sigma, and Aspergillussaitoi a-mannosidase was obtained from Oxford
Alkaline Saponification of Ester-like Bound Fatty Acids-Malarial
Glycosystems.Ionexchange resins were analytical grade and pur-
chased from Bio-Rad. Allsugar andlipid standards were obtained from glycolipids were dried and saponified in 200 pl of 0.05 N KOWMeOH
Sigma. All solvents were analytical or high performance liquid chroma- (1:l; v:v) for 4 h a t 100 "C. The reaction was terminated by drylng the
tography grade. reaction mixture in a Speed-Vac evaporator. After phase partition be-
Parasite-The I! falciparum strain FCBR was obtained from Dr. B. tween water and water-saturated l-butanol, the cleavage rate was es-
Enders, Behring Co., Marburg, Germany. It was maintained in RPMI timated by liquid scintillation counting of aliquots of the organic and
1640 medium supplemented with 25 m Hepes, 21 m sodium bicar- aqueous phases.
bonate, 0.37 m hypoxanthine, 0.1 mg/ml neomycin, and 10% defibri- Enzymatic Cleavage of Glycolipids-The radiolabeled glycolipid mix-
nated (v/v) human A+ fresh frozen plasma (modified from Trager and tures or TLC-purified glycolipids were dried and redissolved in deter-
Jensen (1976)).The cultures contained human A+ erythrocytes (leuko- gent-containingbuffer forthe corresponding enzymatic reactions. Phos-
cyte-depleted erythrocyte concentrates, Blood Donation Center, Univer- phatidylinositol-specific phospholipase C (PI-PLC) digestions were
sity of Marburg, Germany) at 5% hematocrit and were incubated at camed out in 100 plof 0.1 M Tris-HC1(pH 7.4), containing 0.1% sodium
37 "C in gas-tight containers (modular incubation chamber, Flow Labo- deoxycholateand 1unit of B. cereus PI-PLC(BoehringerMannheim). In
ratories) being gassed with a mixture of 90% nitrogen/5% oxygen/5% some cases, TLC-purified glycolipids weretreated with ammonia (32%
carbon dioxide. Development and multiplication of the cultures were NHJMeOH (l:l, v/v); 1h, 37 "C)prior to PI-PLC digestion(Mayoret al.,
followed by microscopicevaluation of Giemsa-stained smears. Synchro- 1990). This treatment is supposed to selectively cleavepalmitic acid at
nous development of the parasite was achieved by the sorbitol method the inositol ring. Glycosylphosphatidyl-specific phospholipaseD (GPI-
(Lambros and Vanderberg, 1979). PLD) treatment was performed under identical conditions, but in this
Metabolic Labeling of l? falciparum Dophozoites-About 5 x lo9 case the incubation buffer contained 2 m CaCl,; 10 pl of normal rabbit
parasite-infected erythrocytes (10% parasitemia) were incubated with serum was used as a source of GPI-PLD (Davitz et al., 1988). Phospho-
250 pCi/5 ml of radiolabeled precursors. Labeling was performed in lipase A, (PLA,) reactions wereperformed in the same incubation
glucose-free RPMI medium (Amimed, Muttenz, Switzerland) supple- buffer but with 1m CaClz and 50 units of the bee venomenzyme. After
mented with 10 m fructose (Schwarz et al. 1986), 25m Hepes, 21 m incubation for 14 h (PI-PLC, PI-PLA,) or 3 h (GPI-PLD) at 37 "C, the
sodium bicarbonate, 0.37 m hypoxanthine, 0.1 mdml neomycin, 0.1 reactions were terminated by heating at 100 "C for 1 min, and the
l l l ~methionine, 2 m glutamine, and 11m glutathione. Radioactively reaction products were extracted twice with 100 pl of water-saturated
labeled fatty acids were coupledto defatted bovine serum albumin at a 1-butanol. The radioactivity in aliquots of the aqueous and organic
molar ratio of 1:l. my~-[~H]Inositol labeling was performed in RPMI phases were counted. The organic phases were analyzed by TLC using
(Life Technologies Inc.Selectamine kit) without vitamins. All labeling solvent system B.
experiments were carried out at 37 "C for 1-2 h. Controllingthe viabil- Nitrous Acid Deamination-Glycolipid mixtures or TLC-purified gly-
ity of the parasites by microscopy of Giemsa-stained blood smears, we colipids weredried and resuspended in 100 pl of freshly prepared 0.1 M
observed that the parasites are viable during the labeling period but die sodium acetate (pH 4.01, containing 0.05% SDS, 0.25 M NaN02, and
if the labeling time is prolonged for more than 4 h. For some experi- incubated for 14 h at ambient temperature. The reaction was termi-
ments, labeling was camed out in the presence of 1pdml tunicamycin. nated by the addition of 5 pl of 6 N HCI and extracted as described above.
Extraction and Purification-Washed parasites were harvested by The organic phases were analyzed on TLC. The radioactivity released
saponin lysis and centrifugation at 4 "C (Gomanet al., 1982).Less polar
into the aqueous phases was analyzed on Bio-GelP4 as described below.
glycolipids wereextracted twice with 2 ml of chlorofodmethanol (CM
Bio-Gel P4 Gel Filtration Analysis-Water-soluble products gener-
2:1, v:v) per lo9 parasites to remove neutral lipids and phospholipids.
ated by enzymatic or nitrous acid treatment of radiolabeled glycolipids
This extraction was camed out for 30 min at room temperature under
were sized on Bio-Gel P4 columns (1 x 130 cm, -400 mesh) in 0.2 M
sonication in a water bath sonicator (Branson 3200, 47MHz). The
ammonium acetate, 0.02% sodiumazide (Masterson et al., 1989). Frac-
residual pellet was then extracted twice with 1 ml of chlorofod
tions (about 850 pl) were collected a t a rate of 1fraction per 24 min.
methanovwater (CMW 10:10:3, v:v:v)to obtain the more polar glycolip-
Glucose oligomers frompartially hydrolyzed dextran were included as
ids. The CM extracts were pooled and subjected to repeated Folch wash-
internal standards and detected by oxidation with 2 mg/ml orcinol in
ing (Sharma et al., 1974).The CMW-extracted glycolipids weredried in
a Speed-Vac (Savant Inc.), and the dried residue was partitioned be- concentrated sulfuric acid.
tween water and water-saturated l-butanol. The organic phase is Generation and Analysis ofCore Glycans from Glycolipids-TLC-
termed "butanol component." purified glycolipids were dephosphorylated, deaminated, and reduced
Preparation of GPI Standards from II: brucei-Ttypanosomes (Zbru- according to a modification of the method described byMayor and
cei MIT variant clone 118) were purified from infected rat blood and Menon (1990).Briefly, the glycolipids were dephosphorylated by treat-
labeled with [3Hlglucosamine, [3H]mannose,or PHlmyristic acid (Me- ment with ice-cold 48% aqueous HF for 60 h at 0 "C. After neutraliza-
non et al., 1988).Alternatively, washed preparations of lysed trypano- tion with frozen saturated LiOH, the reaction mixture was desalted,
somes were labeledwith G D P - ~ - [ 2 - ~ H ] m a ~(2
o spCi)
e or UDP-D-N-[~- deaminated by HNO, treatment for 3 h, and reduced by sodium boro-
3H]acetylglucosamine (2 pCi) (Masterson et al. 1989). All labeling hydride for 5 h. The reduced material was desalted on a tandem ion
experiments were camed out in the presence of tunicamycin. After the exchange columnand filtered through a 0.4-pm and 0.25-w filter. The
labeling, GPI-anchor precursors P2 and P3 (named glycolipid A and C desalted and filtered core glycans were analyzed byDionexHPAEC
by Krakow et al. (1986)) and their biosynthetic intermediates were using gradient elution program 1 (Mayor and Menon, 1990).
obtained by sequential extraction and purified by TLC analysis as de- Anion Exchange HPAECAnalysis of Labeled Glycans-Desalted gly-
scribed by Menon et al. (1988). cans were analyzed by anion exchange chromatography on a Dionex
Solvent Systems forTLC-The solvents for TLC were as follows: A, Basic Chromatography System. The separation was accomplished by
chlorofodmethanolcetic acidwater (25:15:4:2, by volume);
B, gradient elution (program 1)using a Carbopak"?" PA1 (4 x 250 m m )
chlorofodmethanovwater (10:10:2.5, by volume); C, chlorofod column. Monosaccharides were analyzed using isocratic conditions (pro-
 Plasmodium
falciparumin
Glycosylphosphatidylinositols 2599
gram 2). Mono- and oligosaccharide standards were detected by pulsed
amperometric detection. The elution programs for DionexHPAEC were:
1)100% bufTerA(0.1 M NaOH), 0% buffer B (0.1 M NaOH, 0.25 M NaOAc)
up to 6 min after injection, then an increase of buffer B to 30% at 36
min, at a flow rate of 1d m i n and 2) isocratic 15 m~ NaOH at a flow
rate of 1d m i
n .
Exoglycosidase Digestionof Glycolipids-For mannosidase digestion,
TLC-purified glycolipids were deaminated and desalted as described
above. The resulting water-soluble material was resuspended in 100 pl
of 50 m~ sodium acetate (pH 4.5) containing 0.2 m~ ZnClz,0.02%
sodium azide, and 1.7 units of jack bean a-mannosidase and incubated
at 37 "Cfor 24 h. Additional enzyme (0.85 unit) was added 14 h &er the
beginning of the incubation. For A. saitoi a-mannosidase digestion of
dephosphorylated, deaminated, and reducedglycolipids, the glycan
samples were resuspended in 30 pl of 0.1 M sodium acetate (pH 5)
containing 10 microunits of A. saitoi a-mannosidase and incubated for
14 h at 37 "C.
All digestions were stopped by heating a t 100 "C for 1 min. The
reaction mixtures were desalted, filtered, and analyzed by Bio-GelP4 or
by Dionex HPAEC chromatography.
Acetolysis ofNeutra1 Glycans-Radiolabeled glycans obtained via de-
phosphorylation,deamination, and reduction of labeled glycolipids were
cleaved by a modification of the method describedby Mayorand Menon
(1990). This procedure preferentially cleaves Manal4Man linkages
(Rosenfeld and Ballou, 1974). Briefly,the glycans were dissolvedin 30
pl of acetic anhydriddpyridine (l:l, v/v) and incubated for 30 min at
100 "C, to peracetylate. After drying, the samples were resuspended in
50 pl of acetic acidlacetic anhydride/HZSO4(lO:lO:l, v/v) and treated for
6 h a t 37 "C.After neutralization, the samples were partitioned between migration (cm)
250 pl of chloroform and 1 mlof water. The chloroform phases were FTG. 1. TLC analysis of the PHIMan- and [SHIGlcN-labeled gly-
dried and resuspended in 200 pl of methanol/32%ammonia (1:l. v/v) for colipids extracted from I? falciparum. Asexual, intraerythrocytic
16 h at 37 "C to de-0-acetylate the glycans. The de-0-acetylated mate- stages of the malaria parasite P. falciparum were metabolically labeled
rial was desalted and analyzed by Dionex HPAEC chromatography. for 2 h with l3H1Man and L3H1GlcN. Glycolipids were extracted and
Neutral Glycan Standards-Glucose oligomer standards were pre- chromatographed on Silica Gel 60 plates using solvent system B. Ap-
pared according to Yamashita et al. (1982)by partial acid hydrolysisof proximately 10,000cpm were analyzed in each lane. Radioactivity was
Dextran (Sigma). For oligosaccharide analysis onBio-Gel P4 and scanned using a TLC-scanner (Berthold LB 2842). Six L3H1Man and
Dionex HPAEC, these glucose oligomers wereincorporated as internal L3H1GlcN glycolipids were found and named Pfgl e, Pfgl p, Pfgl y, Pfg? 6,
standards. Neutralglycan analysis on Dionex HPAECwas correlated by Pfgl E , and Pfgl I). One very hydrophobic glycolipid labeled only m t h
internal dextran standardsto the elution position of the neutral glycan 13Hl-Manwas identified as dolichol(?)-phosphate-mannoseand there-
portion of radiolabeled intermediates in GPI-anchor biosynthesis of I: fore designated Dol(?)-P-Man.Two with only 13H]GlcN-labeled glycolip-
ids were named Pfgl ( and Pfgl 8. The glycolipids marked with * were not
brucei and the Thy-1 GPI-anchor, prepared by dephosphorylation,
further characterized. 0, origin; F,front.
deamination, and reduction as described above.

RESULTS by Dieckmann-Schuppert et al. (1992b1, using labeling medium

Extraction and Analysis of Glycolipids-We metabolically la- supplemented with 10%human serum.
beled trophozoites ( 3 3 4 3 h after infection), an intraerythro- Additional radiolabeling experiments were performed in or-
cytic, asexual stageof I? falciparum, withdifferent radioactive der to define the composition of these glycolipids. In addition to
precursors, such as L3H1GlcN and [3HlMan. The presence of labeling with mannose and glucosamine, the two most hydro-
tunicamycin had no effect on the glycolipid pattern irrespective philic glycolipids, P f g l a and Pfgl /3,could also be labeled with
of the radioactive precursor used (Gerold et al., 1992b; Dieck- L3Hlmyristic acid, L3Hlpalmitic acid,[3Hlethanolamine, and
mann-Schuppert et al., 1992a). Parasites were purified and my~-[~H]inositol, as expected for glycosylinositol lipids (Table
extracted as described under "Experimental Procedures." Un- I). Labeling of the parasites with L3H1ethanolamine or myo-
infected erythrocytes showed no (<<1%) incorporation of radio- L3H1inositol resulted in more than 98% incorporation of radio-
activity into glycolipids. TLC analysis of the "Fo1ch"-washed activity into phosphatidylethanolamine or phosphatidylinosi-
CM extracts and the butanolcomponent of the CMW extracts tol, respectively (Gerold et al.,1992b; Dieckmann-Schuppert et
(butanol phases) showed qualitatively identical glycolipid pat- al., 1992b). No incorporation of galactose or fucose into ma-
terns inTLC systems A and B. Therefore, the Folch-washed CM larial glycolipids was observed.
extracts and the butanol components werepooled and analyzed Analysis of the Radioactivity Incorporated into the Glycolip-
together usingsolvent system B (Fig. 1).TLC analysis of these ids-Acid hydrolysis productsof the neutralglycans of malarial
pooled glycolipids (termed glycolipid extract) revealed eight glycolipids labeled with L3H1GlcN or L3HlMan were analyzedon
and seven glycolipids labeled with [3H]GlcN (Fig. lA) and Dionex-HPAEC using elution program 2. The analysis showed
L3H1Man (Fig. 1B), respectively. The relative migration (R,) that the incorporated radioactivity was not metabolically con-
values of these glycolipids were 0.25 (Pfgl a),0.33 (Pfg, p), 0.50 verted (more than 98% of the radioactivity is detected as glu-
( P f g ~Y),0.56( P f g ~6), 0.62(Pfgl e), 0.66 (Pfgl J), 0.71 ( P f g l 7),0.85 cosamine or mannose, respectively), in contrast to the results
(Pfgl 01, and 0.9 (dolichol(?)-phosphate-mannose)2(Table I). We described for Toxoplasma gondii GPIs (Schwarz and Tomavo,
have used infected erythrocytes rather thanisolated parasites 1993).
for the following experiments. The glycolipid pattern is con- Identification of most Glycolipids as GPZs-To investigate
served throughout all experiments. However, we have observed whetherthe [3HIGlcN- or L3H1Man-labeled glycolipids are
differences in the relative amount of labeling of single malarial GPIs, we tested the sensitivity of the glycolipid extracts and
glycolipids in various labeling experiments (up to 30 times). also of the single TLC-purified glycolipids toward PI-specific
The obtainedglycolipid pattern is different from that described phospholipase C (PI-PLC)before and &r mild alkaline treat-
ment, the sensitivity toward GPI- specific phospholipase D
(?) indicates that chain length and saturation are unknown (GPI-PLD) and nitrous acid (HN02) treatment (Table I). &r
2600 Glycosylphosphatidylinositols
Plasmodium
in falciparum
I
TABLE
Characterization of malarial glycolipids
Parasites were labeled with C3H1GlcN,L3H1Man, or [3Hlethanolamine,and glycolipids were sequentially extracted with CM and CMW. TLC-
purified glycolipids were subjected to different treatments. After butanol-water phase separation,cleavage was measured
by scintillation counting.
The results summarizedinthis table correspond to the average of two ([3Hlethanolamine)and four (C3H1Man,I3H1GlcN)radiolabelingexperiments.
N L , not labeled; +, sensitive; -, insensitive.
Labeled via Sensitivity to Susceptibility to
Glycolipid
species Proposed structure R, values
[3HlGlcN 13H1Man L3H1EtN PI-PLC +p3Lc
PLD
PLAz HNOz NaOH

EtN-P-Man,-GlcN-Ino(x)-PA 0.25 L L L + f + +
EtN-P-Man,-GlcN-Ino(x)- P A 0.33 L L L + f + +
Man,-GlcN-Ino(x)-PA 0.50 L L NL + f + +
Man,-GlcN-Ino(x)-PA 0.56 L L NL + f + +
Man,-GlcN-Ino (x)-PA 0.62 L L NL + f + +
GlcN-Ino-PA 0.66 L NL NL + + + +
Man,-GlcN-Ino ( x )-PA 0.71 L L NL + * + +
pf; e
(Dol(?)P-Man)
GlcN-Ino( x )-PA 0.85
0.90
L
NL
NL
L
NL
NL
+
"
* +
-
+
-

the treatments, reaction mixtures were partitioned between ring (Menon et al., 1988). Pfgl 6 is totally susceptible to PIA2
water and water-saturated1-butanol. The organic phases con- treatment, consistent with the assumption that this glycolipid
tained uncleaved glycolipids or lipid products. They wereana- has no modification at the inositol.
lyzed by TLC (Fig. 2, left panels), The corresponding aqueous One [3Hlmannose-labeled lipid was shown to be insensitive
phases contained glycan fragments andwere analyzed on Bio- to PI-PLC, GPI-PLD, and HNOZ (Table I). This glycolipid was
Gel P4 (Fig. 2, right panels).Control reactions were performed identified as Dol(?)-P-Man by the following observations: 1)
in parallel. As shown in Fig. 2 (left panel ), only glycolipid 6 is mild acid hydrolysis (2 N HCV1-propanol, 1:1,v/v, 50 "C, 15
cleaved by PI-PLC. All other glycolipids are not susceptible to min) quantitatively released mannose and 2) co-migration on
PI-PLC treatment. To ascertain whether the insensitivity of the TLC (solvent systems A and B) with Dol-P-Man from 2: brucei
other malarial GPIstoward PI-PLC was dueto the presence of and l? falciparum Dol(?)-P-Man synthesized in a cell-free sys-
palmitoylated inositol, as it is described for some GPIs of ?: tem prepared from asexual intraerythrocytic stages of l? fal-
brucei (e.g. P3) and several mammalian GPIs (for review, see ciparum (Gerold et al., 1992a).
Ferguson (1991) and Englund(1993)), the glycolipids Pfgl a,p, After PI-PLC, GPI-PLD, or HNOz treatment of malarial gly-
y, 6, E , q, and 8 were subjected to mild alkaline hydrolysis by colipids, the released oligosaccharide moieties were analyzed
ammonia prior to PI-PLC treatment. Only glycolipid 8 became on Bio-Gel P4 (Fig. 2, right panel). In the cases of GPI-PLD and
45% susceptible, whereas theglycolipids Pfgl a,p, y, 6, E , and q HN02,five peaks with different apparent sizes (depending on
remained insensitive (Table I). the cleavage method) were obtained. In contrast, theradioac-
The glucosamine-inositol fragment of Pfgl a, generated by tivity released by PI-PLC elutes as one peak on the Bio-Gel P4
dephosphorylation and treatment with jack bean a-mannosi- columns. This is consistent with the observation that only Pfgl
dase, was completely (>go%) recovered in the organic phase 6 disappeared from the organic phases afterPI-PLC treatment.
after phase partition. Therefore, Pfgl a (and presumably all Determination of the Size and Presence of Terminal Manno-
other PI-PLC-resistant malarialglycolipids) carry a hydropho- sidase-blocking Residues-The presence of the terminal etha-
bic modification at the inositol ring. nolamine in GPI-anchor precursors renders the structure re-
The treatment of the glycolipids with GPI-PLD and HNOz sistanttojackbeana-mannosidase which requiresan
leads to recovery of >90% (only ~ 1 0 %in the case of Pfgl 6 ) and unsubstituted terminal mannose residue (Li and Li, 1972). Gly-
60%of the radioactivity, respectively, in the organic phases. colipids Pfgl a and p appear to contain ethanolaminesince they
The comparison of the GPI-PLD-generated hydrophobic frag- can be radiolabeled with [3H]ethanolamine. To determine
ments of Pfgl p and the putativeGPI-anchor precursor P3 of .'2 whether this ethanolamineblocking is the terminal mannose in
brucei in the TLC system C showed significant differences in glycolipids Pfgl a and p and whether the other malarialglyco-
the chromatographic behavior (Rf = 0.07 and Rf = 0.75, respec- lipids carry blocking residues, the lipids were tested for their
tively) although both GPIs share the same hydrophilic moieties susceptibility toward jack bean a-mannosidase. TLC-purified
(see below). [3H]Man-labeled Pfgl a, p, y , 6, E, and q and the r3H1GlcN-
However, after HN02 treatment, we found one major and labeled Pfgl 6 and 8 were treated with HN02.The released
several minor productsin theorganic phases.The generationof oligosaccharides were sized on Bio-Gel P4 before and after
alternative deaminationproducts appears to be a common fea- treatment with jack bean a-mannosidase (Fig. 3, A and B ) .
ture of nitrous acid deamination (Krakow et al., 1986; Mayor et HN02-generated fragments of Pfgl a and p (corresponding to
al., 1990). Based on the results that theefficiency of deamina- 7.7 and 6.7 glucose units, respectively; Fig. 3 A , panels A and B 1
tion in our experiments is similar to ofthat the deaminationof are only partly sensitive (Pfgl a;Fig. 3A, panel A1 or insensi-
the GPI-anchor precursors P2 andP3 of ?: brucei, we conclude tive (Pfgl 0; Fig. 3 A , panel B1 ), respectively. The HN02-gener-
that the malarialglycolipids are sensitive to deamination, in- ated and a-mannosidase-treated fragmentsof Pfgl a elute cor-
dicating that they have a nonacetylated hexosamine, as is de- responding to 6.7 and 0.9 glucose units. This indicatedthat Pfgl
scribed for GPIs. a possesses one additional a-mannose side chain which is not
Treatment of the malarialglycolipids with phospholipase A2 blocked like the other mannosyl residues ofPfgl a and are.
(PLA2) leads to the formation of lyso-forms of each malarial The larger fragments of Pfgl a and p produced by jack bean
glycolipid with theexception of dolichol(?)-phosphatee-mannose a-mannosidasetreatment co-elute on Bio-Gel P4withthe
(Dol(?)-P-Man; Table I). However, only about 3 0 4 0 % of each HN02-generated fragmentof the structureethanolamine-phos-
glycolipid, except P f g l 6, was converted to its lyso-form, consist- phate-mannose3-anhydromannoseof the T brucei precursors
ent with the results described for I: brucei GPI-anchor precur- P2 and P3 (6.7 glucose units). The peak intensities of the
sor P3, which was described to have a palmitoylated inositol a-mannosidase-generated fragments of Pfgl a (Fig. 3A, panel
 Glycosylphosphatidylinositols in Plasmodium fakiparum 2601

PI-PLC

0
a
3

migration (cm) Fraction number

FIG.2. Characterization of the [SH]GlcN-labeledglycolipids.[3HlGlcN-labeled glycolipids were treated with PI-PLC, GPI-PLD,and HNOp
as described under “Experimental Procedures.”ARerincubation, the reaction mixture was partitioned between water and water-saturated butanol.
The organic phases were analyzed on silica TLC plates using solvent system B (left panel ). The aqueous phases were analyzed on Bio-Gel P4
columns (right panel) with internal dextran standards (see “Experimental Procedures”).Positions of the standards are shown at the top. The
glycolipids marked with * were not further characterized.

A1 ) show that theside chain mannose is preferentially labeled. acterization, the L3H]GlcN-labeled glycolipidsPfgl a , p, y , 6,E , (,
The HN02-generated fragmentsof the [3Hlmannose-labeled q,and 0 were dephosphorylated by treatment with aqueous HF.
glycolipids Pfgl y , 6 , E , and q (which elute at 4.7,3.7,2.7, and 1.8 This procedureselectivelycleaves phosphodiester linkages.
glucose units, respectively; Fig. 3B) are sensitive to a-manno- The HF-generated glycan fragments were deaminated and re-
sidase treatment (i.e. elution at 0.9 glucose unit afbr treat- duced (as described under “Experimental Procedures”), then
ment), confirming that their terminal mannosyl residues are analyzed by Dionex-HPAEC using elution program 1(Fig. 4, A
not blocked. and B).
The glycolipids Pfgl ( and Pfgl 8, labeled only with L3H]glu- The neutral glycans derived from glycolipids Pfgl a and y
cosamine, are not affected by digestion with jack bean a-man- (Fig. 4A, panels A and C ) co-eluted with a Man4-2,5-anhydro-
nosidase (Fig. 3B, panels F and HI. Together with the failure to mannitol (AHM) standard generated from the Thy-1 GPI-an-
label these two glycolipidswith [3Hlmannoseand theirelution chor (a generous gift from Dr. M.Ferguson). ARer treatment of
size (1.2 glucoseunits), these resultsindicate that Pfgl 5 and 0 the neutralglycans of P f,,a and y with A. saitoi a-mannosidase
do not contain mannose residues. (an exo-mannosidase specific for Manal-2Man linkages), the
Based on the labeling experiments with L3Hlethanolamine, resulting fragments were analyzed on the Dionex-HPAEC sys-
the sensitivity toward dephosphorylation with aqueous HF (see tem and found to co-elute with a Man2-AHMstandard gener-
below), the a-mannosidase digestions, and theconserved struc- ated from I: brucei (Fig. 4A, panels A1 and C1 ). This indicates
ture of other GPI-anchor precursors indicate that theterminal the removal of two al-2-linked mannoses from the glycans of
mannose residues of Pfgl a and p are blocked by ethanolamine- P f g l a and y.
phosphate. Recent experiments show that themalarial surface proteins
Analysis of the Glycans by Dionex-HPAEC-For further char- MSPl and MSP2 possess GPI-anchors with the same neutral
2602 Glycosylphosphatidylinositols
Plasmodium
falciparum
in

A P.I. gl P.I. gl p P.I.gl y

(L P.I. gl 6

"0
lP.B.!. ?.? . 'p I?.?.!. t . ?. "0 I?.!.?. ? .?
200

IO0
R
E
.....,.. .. ...... .. . . ....... ., . ........ .
" 1
*
200

I
IO0

0
45 55 65 75 e5 95 105
hlUdJ"
45 55 6 5 7 5 U5 95 105

Fraction number

B P.f. gl t P.f. 01 c P.I. gl 11 P.f. gl 6

o_

...... . . . 3
D
D
3

5
Fraction number
FIG.3. A, Bio-Gel P4 chromatography of HN02-generated hydrophilic moieties before and after jack bean a-mannosidase treatment. TLC-
purified, [SH]Man-labeled glycolipids Pf,,a, Pf,,p, FYgl y , and Pf,8,were deaminated, and an aliquot (1000-1500cpm for each control;A-D) of the
HNOz-generatedhydrophilic fragment was loaded ona Bio-Gel P4 column. A second aliquot (1500cpm) was incubated with jack bean a-manno-
Ridase and analyzed on the same column (panels A,-&). Glucose oligomers were added to the samples as internal standards, and theirelution
positions are indicated on the top of each profile. Radioactivity(y-axis)in the fractions (850111) was plotted against the fraction number. B,Bio-Gel
P4 chromatography of €€NO,-generated hydrophilic moieties before and after jack bean a-mannosidase treatment. TLC-purified, ISH1Man-labeled
glycolipids Pf,,< and Pf,, and [SH]GlcN-labeledFY,, and Pfzl 8 were deaminated, and an aliquot (1000-1500 cpm for each control; E-H) of the
HN0,-generated hydrophdic fragment was loaded on a Bio-Gel P4 column. A second aliquot (1500cpm) was incubated with jack bean a-manno-
sidase and analyzed on the same column (panels E1-HI).Glucose oligomers wereadded to the samples as internal standards, and their elution
positions are indicated on the top of each profile. Radioactivity (y-axis)in the fractions (850pl) was measured and plotted against the fraction
number.

glycan as Pfgl a.3 dard (Fig. 4A,panels BI and D l ), confirming the removal of one
The neutral glycans of the glycolipids Pfgl p and 6 both co- terminal al-2-linked mannose residue, as described for the
eluted with a Man,-AHM standard prepared from I: brucei neutral glycan of the T brucei GPI-anchor precursors P2 and
(Fig. 4.4, panels B and D ) . When the malarial neutral glycans P3 (Mayor et al., 1990).
were treated with A. saitoi a-mannosidase, the resulting frag- The HF-generated, deaminated, and reduced glycan frag-
ments co-eluted onDionex-HPAEC with a Man2-AHM stan- ments from Pfgl e and q co-eluted with Man2-" and Manl-
AHM standards, respectively (Fig. 4B, panels E and G ) . Con-
3 p. Gerald, L. Schofield, and R. T. Shwarz, manuscript in prepara- sistent with the linkages in the Co~esPonding GPI-anchor
tion. biosynthesis intermediates from I: brucei, the two malarial
 Glycosylphosphatidylinositols in Plasmodium falciparum 2603

A
P.f.gl a P.I. gl p P.t. gl 7 P.I. gl 6

150

s
100. s
50

0. . .
0 10 20 30 50
40 EO 0 10 20 30 40 50 EO 0 10 20 30 40 50 EO 0 10 20 30 40 50 EO

Fraction number

B
P.f. gl E P.f. gl 5 P.f. gI ll p.f. pl e

6
2
1
LOO ~ 2

E .
a
u . .. .. . . . . .
,. . . . .. , , ,. . . .
R
v

I
'Ell
2oo -

150. L
P)
El
100 -
3
tr

50 -E
0 . . . . .
6 , "., ~ 1

IO 20 30 40 50 60 0 10 20 30 4 0 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60

Fraction number
FIG.4. A, Dionex-HPAEC analysis of the HF-generated, dephosphorylated, deaminated, and reduced core glycans. TLC-purified, L3H1GlcN-
labeled glycolipidsPf,,01,Pf,,p, REI7, and Hgl6 were dephosphorylated,deaminated, and reduced as described under "Experimental Procedures."
The neutralglycans were desalted and divided into aliquots. One aliquot (1200-1500 cpm) of each sample was analyzed by Dionex-HPAEC using
gradient elution program 1(profiles A-D). A second aliquot (1500 cpm) was treated with A. saitoi ul-2-specific mannosidase and desalted prior
to the analysis on Dionex-HPAEC(Al-&). The standards indicated on the top of each profile are generated from Thy-1 GPI-anchor and GPI-anchor
precursors of I: brucei. The flow rate was 1 d m i n , and fractions were collected every 0.4 min. B , Dionex-HPAEC analysis of the HF-generated
dephosphorylated, deaminated, and reduced core glycans. TLC-purified, [3HlGlcN-labeled glycolipidsPfgl B , P f,,5, Pfgl q, and H,,8 were dephos-
phorylated, deaminated, and reduced as described under "Experimental Procedures." The neutral glycans weredesalted and divided into aliquots.
One aliquot (1200-1500 cpm) of each sample was analyzed by Dionex-HPAEC using gradient elution program 1 (profiles E-H). A second aliquot
(1500cpm) was treated with A. saitoi ul-2-specific mannosidase and desalted prior to analysis on Dionex-HPAEC(El-Hl). The standards indicated
on the top of each profileare generated from Thy-1 GPI-anchor and GPI-anchor precursors of I: brucei. The flow rate was 1d m i n , and fractions
were collected every 0.4 min.

glycans were found to be resistant to A. saitoi a-mannosidase labeled by [3H]mannose, Fig. L4)showed that both fragments
(Fig. 4B, panels E l and G1). were identical and co-eluted with 2,5-anhydroma~itol(Fig.
Dionex-HPAEC analysis of the neutralglycans derived from 4B, panels F and H ) . Treatment with A. saitoi a-mannosidase
the [3H]GlcN-labeled glycolipids PfBl& and
' 0 (which were not had no effect on the elution behavior of the glycans of both
 2604 Glycosylphosphatidylinositols in Plasmodium falciparum
P.f.g1 (1 P.i.gl p P.1.91 -f

250 -g3.'4:- .. . . .
aP
I
200

I50

FIG.5. Dionex-WAECanalysis of 100

L
thedephosphorylatedcoreglycans
prior to and afterjack bean a-manno- 50
sidase and acetolysis treatment.TLC-
purified, [3HlGlcN-labeled glycolipids
Pfgl 0
a, Pfgl p, and FYgl y were dephosphory-
lated, deaminated, and reduced. The de-
salted neutral glycans were divided into 200
aliquots. The first aliquot (1200-1500
cpm) wasanalyzed by Dionex-HPAECus- I50
ing elution program 1 (profiles A X ) . A E
second aliquot (1200-1500 cpm) was di-
gested with a-mannosidase from jack
bean. The desalted fragments were also
3 100

analyzed on Dionex-HPAEC (A,C1). The

third aliquot (3000 cpm) was subjected t o 50
acetolysis and analyzed on Dionex-
HPAEC (A+?& using the same elution .. . . .
.. .
0
program. The standards indicated on the
top of each profile are generated from
Thy-1 GPI-anchor and GPI-anchor pre- 200
cursors of I: brucei. The flow rate was 1
mumin, and fractions were collected every
0.4 min. 150

IO0

50

0
IO 20 30 40 50 60

Fraction number

glycolipids, indicating that they have no terminal al-2 man- produce two putative glycosylphosphatidylinositol membrane
nose residue (Fig. 48,panels F l and HI ). anchor precursors ethanolamine-phosphate-6(al-2Man)
JackBeana-MannosidaseDigestion of NeutralGlycans Mancul-2Manal-GMana1-4GlcN-PI (Pfgl a) and ethanola-
-Digestion of HF-generated neutral glycans of the glycolipids mine-phosphate-6Manal-2Mana1~Mana1-4GlcN-PI (Pfgl p)
Pfgla,Pfgl p, and Pfgl y with jack bean a-mannosidase produced and a series of minorglycosylphosphatidylinositols (GPIs)
single glycans for each malarial neutral glycan (Fig. 5, panels showing a lesser degree of glycosylation.
A l , B l , and C1 ). These single glycans co-elute in each case with The glycolipid extracts contain Dol(?)-Man,six f3H]Man-and
a 2,5-anhydromannitol standard. These data show that the eight [3HlGlcN-labeled glycolipids which are shown to be GPIs
neutral glycans of Pfgl a,/3,and y consist of a-mannose residues by different chemical and enzymatic cleavages. All glycolipids,
without additional non-mannose components. except Dol(?)-P-Man,are sensitive to GPI-PLD, PLA2, HNO2,
Acetolysis of the Neutral Glycans of Malarial Glycolipids"T0 and NaOH. Only the glucosamine-labeled Pfgl 5 is sensitive to
test for Manal-GMan linkages, we made use of the relatively PI-PLC. All other glycolipids are insensitive to PI-PLC treat-
selective acetolysis conditions described by Rosenfeld and Bal- ment suggesting that these glycolipids might have a modified
lou (1974). The HF-generated deaminated and reduced neutral inositol structure, which is described to hinder PI-PLC cleav-
glycans of [3H]GlcN-labeled Pfgla,Pfgl p, and Pfgl y were sub- age (Roberts et al., 1988). Additional evidence for a modified
jected to partial acetolysis andthen analyzed onDionex inositol structure is provided by the only partial sensitivity of
HPAEC (Fig. 5 , panels A 2 , B2,and C2).Besides the unhydro- the malarial GPIs toward PLA2 which is similar to that of the
lyzed neutral glycans of these three malarial glycolipids, we I: brucei GPI-anchor precursor P3 (Menon et al., 1988,1990b).
found in all cases material co-eluting with Manl-AHM and Different protein-bound GPIs (Roberts et al., 1988; Walter et
AHM standards. The identification of a Manl-AHM fragment al., 1990; Claytonand Mowatt, 1989)and GPI-lipids (Krakowet
generated by acetolysis is consistent with the presence of a al., 1989; Mayor et al., 1990; Field et al., 1991) are described
Manal-GMan bond between the first andsecond mannose resi- which have inositol structures modified by palmitic acid. In
dues of the malarial glycolipids Pfgl a, Pfgl p, and Pfgl y. The these cases, pretreatment with ammonia prior to PI-PLC treat-
observation of under- and overdigestion products in acetolysis ment leads to partial susceptibility toward PI-PLC (Krakow et
studies is confirmed by the results of Mayor et al. (1990). al., 1989; Mayor et al., 1990). Malarial glycolipids, however,
could not be rendered PI-PLC-sensitive by pretreating with
DISCUSSION ammonia. Partition of the glucosamine-inositol-Xfragment of
In this report we provide evidence that the asexual, intra- Pfgla into the organic phase and thedifferent chromatographic
erythrocytic late stages of the malaria parasite P. falciparum behavior of the GPI-PLD-generatedfragment of Pfgl p and the
 Glycosylphosphatidylinositols
in Plasmodium
falciparum 2605
respective fragment of P3 indicate that all malarial GPIs, ex- 4GlcN-PI (Pf,, p ) and Manal-2Mana1-6Manal-tGlcN-PI
cept Pfgl (,carry a hydrophobic modification on theinositol ring (Pf,, 6).
different from palmitic acid which had been described for try- A pathway of reactions in the synthesisof GPI-anchors in-
panosomal P3 (Menon et al., 1988). volving sequential addition of monosaccharides, derived from
We describe two malarial glycolipids with a single glucosa- activated precursors, has been described for T brucei (Master-
mine as their hydrophilic head group (Pfgl ( and e). Both gly- son et al., 1989; Doering et al., 1990; Mayor and Menon, 1990;
colipids differ in their chromatographic behavior and their sen- Mayor et al., 1990; Field et al., 1992; Menon et al., 1993), Toxo-
sitivity toward PI-PLC. Therefore, we postulate a n additional plasma gondii (Tomavo et al., 19921, and some mammalian
hydrophobic modification at the inositol of Pfgl 8. This would be cells (Puoti et al., 1991; Stevens and Raetz, 1991). The Bio-Gel
consistent with a biosynthetic pathway beginning with gluco- P4 and the Dionex-HPAEC analysis of the different hydrophilic
samine-PI (Pf,,() followed by the addition of a hydrophobic fragments of the malarial glycolipids Pfgl c, Pfgl q, Pf,, (, and
modification (Pf,, 0) prior to the additionof the first mannose Pfgl e, in comparison to neutral glycans generated from inter-
residue. A similar biosynthetic pathway hasbeen described for mediates in theGPI-anchor biosynthesis of T brucei, their in-
mammalian GPIs (Urakazeet al., 1992; Hiroseet al., 1992). At sensitivity to A. saitoi a1-2-mannosidase, and the remarkable
this stage, it is difficult to assess the exact location of this conservation of the core glycan on all investigated GPIs leads
modification on the inositol ring, but it is assumed to be an us to propose the structures Manal-GManal-tGlcN (Pf,, e),
ester-bound hydrophobic moiety because of its sensitivity to GlcN (Pf,, (), Manal4GlcN (Pf,, q), and GlcN (Pfgl 0 ) for these
treatment withNaOH. malarial glycolipids. The identification of potential biosyn-
One glycolipid releases mannose upon mild acid hydrolysis thetic intermediatesof GPI-anchor synthesis andtwo putative
specific for Dol-P-Man (McDowell and Schwarz, 1988). This GPI-anchor precursorsin I! falciparum labeled inculture
glycolipid is presumably Dol(?)-P-Man as judged by its chro- which, with one exception, are substitutedat the inositol ring is
reminiscent of the pathways shown for mammalian cells.
matographic properties. It co-chromatographs with a manno-
lipid synthesized in thecell-free system (Gerold et al. , 1992a) In this paper we present preliminary data which suggest
that theinositol ring of malarial GPIs(except Pfgl (1 is modified
showing properties of Dol-P-Man (alkali stability, chromato-
by a hydrophobic residue different from palmitic acid, the only
graphic behavior on DEAE-cellulose), the synthesisof which is
hydrophobic modification on the inositol ring of GPIs described
inhibited in vitro by amphomycin. The donor of the mannose
so far (for review, see Englund (1993)). Especially the hydro-
residues to thecore glycan of l! brucei GPIs hasbeen shown to
phobic elements of malarial GPIs maybe responsible for some
be Dol-P-Man (Schwarz et al., 1989; Menon et al., 1990b).
of the pathological properties of I? falciparum in severe ma-
Therefore, by analogy, a n isoprenoid-P-Man can be postulated
laria. For instance, thenon-protein-bound malarial GPIsPfgl a
to play a similar role for at least some of the mannose residues and Pfgl p are potential inducers of cytokine r e l e a ~ eTogether
.~
of the malarial GPIs. The lack of Dol-PP-(GlcNAc), Dol-PP- with findings that purified GPIs derived from pronase hydro-
(GlcNAc)2,and higherglycosylated intermediates is consistent lysis of MSPl and MSP2 induce cytokine release, transient
with the findings that the asexual, intraerythrocytic stagesof pyrexia, hypoglycemia, and lethal cachexia in mice (Schofield
€? falciparum do not produce detectable amounts of dolichol and Hackett, 1993) and with other evidence (Kwiatkowski et
cycle intermediates as observed in this and previous (Dieck- al., 1990), these resultspoint to an important role of this class
mann-Schuppert et al., 1992a; Gerold et al., 1992a,1992b) of molecules in the pathology of malaria.
studies. Elucidation of the structures and biosynthetic pathways of
We show that €? falciparum produces mannose- and glucosa- malarial GPIs may provide a basis for the development of a
mine-labeled GPIs, whose glycan backbones are mannosylated glycolipid-based “antidisease” vaccine (Playfair et al., 1990;
to a variable degree. The dephosphorylated, deaminated, and Schofield and Hackett, 1993).
reduced neutral glycans analyzed by HPAEC (Dionex) and the Additional studies in progress using a cell-free system will
deaminated hydrophilic fragments analyzed on Bio-Gel P4 of provide more detailed information of the biosynthetic pathway
the different malarial glycolipids co-elute with the respective of malarial GPIs and help to determine whether Pfgl p is an
glycans or hydrophilic fragments of the GPI-anchor of Thy-1 additional possible GPI-anchor precursor. Also, the design and
(hexosaminidase-treated) and the GPI-anchor precursor P2 of testing of inhibitors of GPI biosynthesis shouldbe facilitated by
I: brucei and its biosynthetic intermediates. Labelingwith studying some of the reactions involved in thecell-free system.
f3HIethanolamine and the analysis of the neutralglycans after
HF dephosphorylation strongly suggest Pfgl a and Pfgl p to be Acknowledgments-We thank S. Koslowski, M. Eppinger,and S.
ethanolamine-phosphate-carrying, putative GPI-anchorpre- Kauer for technical assistance and B. Striepen, C. Zinecker, andDr. M.
A. J. Ferguson for helpful discussions. We gratefully acknowledgeDrs.
cursors. The resultsof the linkage type analysisof the terminal A. K. Menon and S. Tomavo for critical reading of the manuscript. We
mannoses in the malarial glycolipids Pfgl a and Pfgl y using would like to thank Dr. V. Kretschmer, Blood Donation Center, Univer-
al-2-specific mannosidase from A. saitoi and acetolysis are sity ofMarburg,forproviding human erythrocytes and Dr. M. A. J.
consistent withP f g l a and y carrying a n additional al-2-linked Ferguson for a generous gift of Thy-1 neutral glycans. P. G. thanks the
Hessische Graduiertenfdrderung for a fellowship and V.G, A. G. andA.
mannose side chain at the terminal mannose. The linkage of B. for continuous support.
the additional mannose residue to the terminal mannose is
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Plasmodium
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in
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