Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI-anchored C-terminal fragments of MSP-1 are recovered in Triton X-100 resista
Trang 1Plasmodium falciparum merozoite surface protein 1
Glycosylation and localization to low-density, detergent-resistant membranes
in the parasitized erythrocyte
Daniel C Hoessli1, Monique Poincelet1, Ramneek Gupta2, Subburaj Ilangumaran3and Nasir-ud-Din4
1 Department of Pathology, Centre me´dical universitaire, Geneva, Switzerland; 2 Center for Biological Sequence Analysis,
Technical University of Denmark, Lyngby, Denmark; 3 Department of Experimental Therapeutics, Ontario Cancer Institute, Toronto, Canada;4Institute of Biomedical Sciences, Pakistan and HEJ Institute of Chemistry,
University of Karachi, Lahore, Pakistan
In addition to the major carbohydrate moieties of the
glycosylphosphatidylinositol (GPI) anchor, we report that
Plasmodium falciparummerozoite surface protein 1 (MSP-1)
bears O-GlcNAc modifications predominantly in
b-ano-meric configuration, in both the C- and N-terminal portions
of the protein Subcellular fractionation of parasitized
erythrocytes in the late trophozoite/schizont stage reveals
that GPI-anchored C-terminal fragments of MSP-1 are
recovered in Triton X-100 resistant, low-density membrane
fractions Our results suggest that O-GlcNAc-modified
MSP-1 N-terminal fragments tend to localize within the parasitophorous vacuolar membrane while GPI-anchored MSP-1 C-terminal fragments associate with low-density, Triton X-100 resistant membrane domains (rafts), redis-tribute in the parasitized erythrocyte and are eventually shed
as membrane vesicles that also contain the endogenous, GPI-linked CD59
Keywords: detergent-resistant membranes; malaria; mer-ozoite surface protein; O-GlcNAc modification; vesicles
In the blood-stage forms of the malarial parasite Plasmodium
falciparum, the merozoite surface protein 1 (MSP-1) is a
major surface component [1] that undergoes selective
proteolytic processing and reassembly in preparation for
erythrocyte invasion [1–4] MSP-1 is linked to the parasite
plasma membrane via a glycosylphosphatidylinositol (GPI)
anchor [5], but the functional consequences of this mode of
anchoring for the merozoite to interact with the erythrocyte
have not been fully evaluated [6] In addition to the
GPI-anchor modification, MSP-1 also contains mono- or
oligo-saccharides in O-linkage to serines or threonines
[7–10] N-linked carbohydrates have also been described in
association with asparagines on MSP-1 [9], despite the
reported lack of N-glycosylating machinery in P falciparum
parasites [11] As P falciparum merozoite maturation takes
place within an intraerythrocytic network of modified
(parasitophorous vacuolar membrane) and newly made
(tubo-vesicular network) membranes [12,13], it is possible
that parasite surface proteins also constitute substrates for
carbohydrate-modifying enzymes of the erythrocyte In
normal erythrocytes, O-GlcNAc modifications of serines/
threonines in intracellular proteins occur in a manner
reciprocal to phosphorylation [14] and O-GlcNAc addition
is considered a widespread and general mechanism for protein modification [15] In this study, we have analysed MSP-1 for the presence of O-GlcNAc-modified serines and threonines, using specific antibodies to map the biosynthe-tically labelled modifications to the N and C terminus of the MSP-1 protein [16] The presence of O-GlcNAc on both the C- and N-terminal ends of MSP-1 was confirmed by exogalactosylation and two-thirds of the [3H]GlcN label incorporated into MSP-1 was sensitive to Jack Bean b-N-acetylglucosaminidase, suggesting the presence of O-GlcNAc moieties in b-anomeric linkage [15] Predictions for a- and b-anomeric O-GlcNAc sites in five known MSP-1 sequences were made using methods based on artificial neural networks which are competent in recognizing fuzzy sequence motifs, and two distinct sets of a- and b-O-GlcNAc sites have been predicted The GPI-anchored 19-kDa C-terminal fragment was found associated with detergent-resistant, low-density membranes of the parasi-tized erythrocyte, suggesting that GPI-linked MSP-1 prod-ucts redistribute within the membrane network of the parasitized host cell aboard detergent-resistant membrane domains Infected erythrocytes were also found to release membrane vesicles containing parasitic 19-kDa MSP-1 fragments and endogenous CD59, both GPI-linked proteins
Materials and methods
Materials Anti-MSP-1 mAbs reactive with the C (3B10) and N terminus (7B2) [16], were obtained from J.A Lyon (Walter Reed Army Institute of Research, Washington, USA) A human immune serum against blood stage antigens was used to detect all MSP-1 epitopes [10] Anti-CD59 mAb
Correspondence to D C Hoessli, Department of Pathology,
Centre me´dical universitaire, 1, rue Michel-Servet,
1211 Geneva 4, Switzerland.
Fax: +41 22 7025746, Tel.: +41 22 7025893,
E-mail: Daniel.Hoessli@medecine.unige.ch
Abbreviations: GPI, glycosylphosphatidylinositol; MSP-1,
merozoite surface protein 1.
(Received 9 September 2002, revised 13 November 2002,
accepted 26 November 2002)
Trang 2MEM-43 was supplied by V Horejsi (Academy of Sciences
of the Czech Republic, Prague) Recombinant, bovine
b-1,4-galactosyltransferase was from Calbiochem
b-galac-tosidase (bovine brain) and b-N-acetylglucosaminidase
(Jack Bean) were from Sigma
Metabolic radiolabelling of M25 ZaireP falciparum
parasites
Asexual blood stage parasites were cultured in the
asyn-chronous mode in 10-mL cultures at 37C in a candle jar
The culture medium contained 5% v/v human group A+
erythrocytes, in RPMI medium supplemented with 10%
ARh+ human serum (HD Supplies, Aylesbury, Bucks,
UK) and 0.1% glucose Labelling was carried out for 16 h
with 50 lCiÆmL)1 D-[6-3H]glucosamine hydrochloride
([3H]GlcN; 40 CiÆmmol)1, American Radiolabeled
Chemi-cals, supplied by Anawa, Wangen, Switzerland), as
previ-ously carried out to show carbohydrate modification of
plasmodial proteins [9,17]
At the end of the labelling time, culture supernatants were
collected and centrifuged at low speed (2000 r.p.m., 5 min)
to remove uninfected and parasitized erythrocytes The
supernatant was centrifuged sequentially at 15000 g for
10 min at 4C and the 15000 g supernatant again at
100 000 g for 30 min at 4C (Beckman SW41 rotor) to
collect the released membrane vesicles The 100 000 g pellet
was analysed as a source of parasite-free membrane
nanovesicles (Fig 1A)
To study the distribution of MSP-1 and its fragments, the
[3H]GlcN-labelled, parasitized erythrocytes at the late
trophozoite/schizont stage were collected by sedimentation
over a 70% Percoll (Pharmacia) gradient [18] and
hemolysed in H2O in the presence of protease inhibitors
(0.19 mMleupeptin, 0.17 mMchymostatin, 2 mM
N-p-tosyl-L-lysine chloromethyl ketone (TLCK), 2 mM
N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 1 mM
phenylmethanesulfonyl fluoride and 1 mM
ortho-phenanthro-line; Fig 1B) A small aliquot (5%) of the [3H]GlcN-labelled
parasites and haemoglobin-free erythrocyte membranes was
directly solubilized in SDS/PAGE sample buffer (see Fig 2,
[3H]GlcN; tot), resolved on SDS/PAGE and the labelled
proteins revealed by fluorography The gel was soaked in
Enlightning (NEN), dried and exposed to Hyperfilms
(Amersham)
The bulk of this material was resuspended in TKM buffer
(50 mMTris/HCl pH 7.4, 25 mMKCl, 5 mMMgCl2, 1 mM
EGTA) containing 1% Triton X-100, 36% sucrose and
protease inhibitors, and centrifuged at 250 000 g in a SW 50
rotor for 16 h at 4C This procedure allows the parasites
to be pelleted with remnants of the parasitophorous
vacuolar membrane [19] (P, Fig 1B), and to be separated
from Triton X-100 soluble and Triton X-100 resistant
components of the parasitized erythrocyte membranes that
are recovered in the supernatant (S, Fig 1B)
SDS/PAGE, immunoprecipitation and Western blotting
In 10% of the supernatant material, the proteins were
precipitated with chloroform/methanol for Western blotting
analysis [20] with anti-(C-ter) or anti-(N-ter) mAbs The
precipitated proteins were separated on a 10% minigel
Fig 1 Subcellular fractionation protocols utilized to generate extracts containing parasite proteins from [3H]GlcN-labelled and unlabelled,
P falciparum-infected erythrocytes (A) Isolation of microvesicles and nanovesicles from [3H]GlcN-labelled, infected erythrocytes (B) Isola-tion of parasite extracts (P) and Triton X-100 extracted proteins (S) from [ 3 H]GlcN-labelled, infected erythrocytes (C) Isolation of Triton X-100 insoluble membranes and Triton X-100 soluble membrane and cytosolic proteins from infected erythrocytes.
Trang 3and transferred to nitrocellulose (Hybond-C, Amersham
Pharmacia Biotech) with a semidry blotting apparatus
(Bio-Rad) After 2 h of blocking at room temperature in
NaCl/Tris/Tween (10 mMTris/HCl pH 7.4, 100 mMNaCl,
0.05% Tween 20) containing 5% low-fat, dry milk powder
(NaCl/Tris/Tween/5% MP), the filters were incubated with
antibodies in NaCl/Tris/Tween/5% MP overnight at 4C
Thoroughly washed filters were incubated with
horseradish-peroxidase-conjugated secondary antibodies for 1 h at
room temperature Chemiluminescence development was
carried out with the Immun-Star Pack reagents (Bio-Rad)
and the filters exposed to X-Omat Kodak films
The bulk (90%) of the supernatant (S, Fig 1B) was
dialysed to remove sucrose and sequentially
immunopre-cipitated with Sepharose 4B-coupled anti-(N-ter), followed
by anti-(C-ter) mAb The antibodies were covalently
coupled to CNBr-activated Sepharose 4B beads according
to the manufacturer’s instructions Incubation with each
mAb was carried out for 6–10 h at 4C on a rotating wheel,
the antibody beads were washed in TKM/Triton X-100
containing protease inhibitors, and the bound antigens
extracted with SDS/PAGE sample buffer The [3
H]GlcN-labelled, immunoprecipitated proteins were revealed by
fluorography, as described above
The parasite pellet (P, Fig 1B) was extracted with 10%
SDS (1 h at room temperature), 10% of the extract sampled
for Western blotting and the remainder diluted with Triton
X-100 and BSA to obtain a final concentration of 0.05%
SDS, 0.5% Triton X-100, 10 lgÆmL)1 BSA, suitable for immunoprecipitation Sequential immunoprecipitation with Sepharose-coupled anti-N followed by anti-(C-ter) mAb was carried out as with the supernatant (S) material and the [3H]GlcN-labelled, immunoprecipitated proteins visualized
by fluorography as described above
Fig 3 Vesicular release of C-terminal MSP-1 fragments by parasitized erythrocytes The [ 3 H]GlcN-labelled, parasitized erythrocytes in the culture were purified over Percoll and processed for immunoprecipi-tation as outlined in Materials and methods Immunoprecipiimmunoprecipi-tation was carried out with Sepharose-coupled 3B10 mAb and human malaria-immune serum coupled to protein A/G beads, and the immunopre-cipitated, labelled bands revealed by fluorography (Whole extract: IP anti C-ter; IP immune serum) The supernatant of [ 3 H]GlcN-labelled cultures at the late trophozoite/schizont stage were allowed to settle and the supernatant collected Remaining erythrocytes were sedi-mented at 2000 r.p.m for 10 min The resulting supernatant was centrifuged once at 16 000 g to remove pelletable parasites, parasitized erythrocytes and uninfected erythrocytes The last supernatant was ultracentrifuged at 100 000 g and the membrane pellet (nanovesicles) was resu spended in TKM One-tenth was extracted with 10% SDS sample buffer (vesicles: unselected) and the [3 H]GlcN-labelled bands revealed by fluorography The remaining 90% was divided into two aliquots: one aliquot was extracted with 10% SDS and diluted to 0.5% Triton X-100, 0.05% SDS, 10 lgÆmL)1BSA for immunoprecipitation with Sepharose-coupled anti-MSP-1 C terminus (vesicles: IP anti-C-ter) and the labelled bands revealed by fluorogra-phy The other aliquot was kept in TKM and incubated for 6–10 h with Sepharose-coupled anti-(C-ter) The immunoselected vesicles were washed in TKM and extracted with SDS/PAGE buffer, trans-ferred to nitrocellulose and probed with MEM-43 anti-CD59 mAb (vesicles: WB anti CD59) and revealed by chemiluminescence.
Fig 2 Both C-terminal and N-terminal fragments of MSP-1
biosyn-thetically incorporate [ 3 H]GlcN The parasitized erythrocytes were
isolated by Percoll gradient centrifugation and lysed in hypotonic
buffer The resulting parasites and membrane ghosts were extracted in
TKM-1% Triton X-100/35% sucrose and ultracentrifuged to yield a
pellet (P) of parasites and a supernatant (S) containing Triton
X-100-resistant complexes and Triton X-100-soluble membrane proteins.
[ 3 H]GlcN: fluorogram of [ 3 H]GlcN-labelled P falciparum proteins in a
total (tot), SDS extract of Percoll-purified parasitized erythrocytes
following hemolysis C-ter and N-ter: probing with the 3B10 (C-ter) or
7B2 (N-ter) mAb following transfer to nitrocellulose and detection by
chemiluminescence (WB), or immunoprecipitation with
Sepharose-bound mAb and fluorography (IP) Results shown are representative
of three separate experiments.
Trang 4One-tenth of the nanovesicle pellet (Fig 1A) was directly
solubilized in SDS/PAGE sample buffer, resolved on a 10%
minigel and processed for fluorography (unselected, Fig 3)
The remainder of the nanovesicle pellet was divided into two
aliquots One aliquot was solubilized in 10% SDS and
subsequently diluted with TKM/Triton X-100 and BSA to a
final concentration of 0.5% Triton X-100, 0.05% SDS,
10 lgÆmL)1BSA The MSP-1 19 kDa C-terminal fragment
was immunoprecipitated with Sepharose-coupled
anti-(C-ter) mAb as described above The bound MSP-1
fragments were eluted in SDS/PAGE sample buffer,
resolved on a 10% minigel and processed for fluorography
The other aliquot was incubated in TKM with Sepharose
4B-coupled anti-(C-ter) mAb for 6–10 h at 4C (rotating
wheel) The antibody-bound membranes were extracted in
SDS/PAGE sample buffer, resolved on a 10% minigel,
transferred to Hybond, probed with MEM-43 anti-CD59
mAb and visualized by chemiluminescence
Exogalactosylation and deglycosylation of MSP-1
Auto-galactosylated, recombinant
b-1,4-galactosyltrans-ferase (20 mU, Calbiochem) was used to probe
nitrocel-lulose-immobilized parasite proteins for nonreducing
terminal GlcNAc residues [21], using UDP-[6-3
H]galac-tose (40 CiÆmmol)1, American Radiolabeled Chemicals)
as galactose donor The MSP-1 proteins were specifically
immunoprecipitated from a 10% SDS extract of
Percoll-purified parasitized erythrocytes This SDS extract was
diluted with Triton X-100 as described above for the
parasite pellets, and incubated with Sepharose-coupled
antibodies Affinity-purified C- or N-terminal MSP-1
proteins were eluted from the solid-phase antibodies with
SDS/PAGE sample buffer, electrophoretically separated
and transferred to nitrocellulose The presence of the
C- and N-terminal fragments was confirmed by probing
a parallel lane containing identically immunoprecipitated
MSP-1 proteins with anti-C and anti-(N-ter) mAbs and
the adjacent nitrocellulose lane containing the
appropri-ate protein was cut and subjected to exogalactosylation
Cut nitrocellulose pieces corresponding to the 195 (whole
1), 56 (C-terminal) and 86 (N-terminal) kDa
MSP-1 proteins (marked by asterisks in Fig 2) were incubated
with 20 mU autogalactosylated, recombinant
galactosyl-transferase overnight at 37C in 0.1M cacodylate buffer
pH 7.2, with 100 lM MnCl2, with 1 lCi UDP-[6-3H]
galactose as galactose donor After washing in 0.1M
citrate-phosphate buffer pH 4.3, degalactosylation was
carried out on the labelled proteins with 10 mU
b-galactosidase in the same buffer Radioactivity in the
exogalactosylated and the degalactosylated bands was
counted in a liquid scintillation counter Control
exoga-lactosylation reactions included anti-C or anti-(N-ter),
sham selected material from lysates of uninfected
eryth-rocytes, and nitrocellulose-transferred BSA, that does not
bear the O-GlcNAc modification and thus cannot be
exogalactosylated
b-N-acetylglucosaminidase from Jack Bean (Sigma) was
utilized to remove biosynthetically incorporated [3H]GlcN
on MSP-1 retained on Sepharose-coupled anti-(N-ter) or
anti-(C-ter) mAbs b-N-acetylglucosaminidase treatment of
MSP-1 bound to antibodies was carried out as described
[22] The radioactivity remaining on the beads after b-N-acetylglucosaminidase or control b-galactosidase treat-ments was counted
Prediction of O-GlcNAc addition sites on MSP-1 protein Sequences for the Ghana-RO33, Png-MAD20, Uganda, Thai-K1 and Wellcome (Swiss-Prot accession no P19598, P08569, P50495, P04932, P04933) isolates were aligned as recommended in [23] using the sequence editor Jalview (M Clamp, unpublished data) Alignment for the C-ter-minal third of the Ghana-RO33 isolate was missing in [23], and this was performed manually O-GlcNAc modified sites
in the a-anomeric configuration were predicted using the
DICTYOGLYC 1.1 prediction server http://www.cbs.dtu.dk/ services/DICTYOGLYC/ [24] and b-anomeric O-GlcNAc sites were predicted using the YinOYang 1.2 prediction server (R Gupta, S Brunak & J Hansen, unpublished data) available at http://www.cbs.dtu.dk/services/YinOYang/ Both prediction methods are based on neural networks and incorporate a surface-accessibility derived threshold which makes it more probable for a predicted site to be on a surface exposed Ser/Thr in the protein The design of these methods is similar to NetOGlyc, a successful predictor for O-GalNAc mucin type glycosylation sites [25] The methods have been rigorously cross-validated and have at least one experimental verification for prediction of each type of linkage DictyOGlyc, the O-a-GlcNAc predictor, was trained on an in vivo set of secreted and membrane proteins
of Dictyostelium discoideum, and the O-b-GlcNAc predictor was trained on a set of intracellular eukaryotic (mostly mammalian) proteins Predictions from the servers were then mapped onto the alignment
Equilibrium sucrose density gradient centrifugation
ofP falciparum-parasitized erythrocytes Lysates of Percoll-purified, late trophozoites/schizonts in TKM/1% Triton X-100 were adjusted to 40% sucrose, placed at the bottom of a Beckman SW41 tube, overlaid with 6 mL 36% and 3.5 mL 5% sucrose in TKM buffer (Fig 1C) Following centrifugation at 250 000 g for 16 h at
4C, 1-mL fractions were collected from the top Equal volumes (50 lL) of the floating, detergent-resistant mem-branes containing GPI-linked proteins (fractions 3 and 4) and the Triton X-100 soluble proteins (fractions 5–10) were concentrated and analysed by Western blotting [20] as described above The parasite pellet containing remnants of the parasitophorous membrane (fraction 11) was solubilized
in SDS/PAGE sample buffer and a matching amount subjected to Western blotting MSP-1 was detected with the anti-(C-ter) mAb and the erythrocyte surface molecule CD59, a GPI-linked complement defence protein, was detected with the MEM-43 mAb
Results
MSP-1 is O-GlcNAc-modified in the N and C termini Fig 2 compares the MSP-1 protein and fragments detected
in extracts of [3H]GlcN-labelled, parasitized erythrocytes by immunoprecipitation or Western blotting The parasites
Trang 5recovered in the pelletable material of the Triton X-100/
36% sucrose extract were contained within residual
para-sitophorous vacuolar membranes [19] The supernatant (S)
contained both the Triton X-100 solubilized proteins and
the Triton X-100 resistant complexes emanating from the
membranes of the parasitized erythrocyte The
fluorograph-ic pattern of [3H]GlcN-labelled proteins from the total SDS
extract of the parasitized, hemolysed erythrocytes is shown
for reference ([3H]GlcN; tot)
The 195-kDa MSP-1 protein was labelled in the total
[3H]GlcN extract and immunoprecipitated by both
anti-(C-ter) and anti-(N-ter) mAbs in the parasite pellet as well as
in the supernatant Western blotting with the anti-(C-ter)
mAb showed a higher ratio of intact MSP-1 to the 19-kDa
fragment in the parasite pellet than in the supernatant
suggesting that MSP-1 C-terminal 19-kDa peptides were
preferentially found in the membrane network of the
parasitized erythrocyte One 100-kDa peptide bearing both
N- and C-terminal epitopes was detected by both antibodies
on Western blots of the pellet and supernatant The 86-kDa,
N-terminal specific peptide was detected by Western
blotting and immunoprecipitated as a [3H]GlcN-labelled
fragment only in the parasite pellet Likewise a further
N-specific and [3H]GlcN-labelled peptide of 40 kDa was
also immunoprecipitated from the parasite pellet
The C-terminal specific peptides consisted of one group
of three bands between 48 and 58 kDa, detectable by
Western blotting and immunoprecipitated as [3
H]GlcN-labelled peptides The other C-terminal peptide of 19 kDa
formed a heterogeneous group of peptides between 10 and
19 kDa (on Western blot) and predominated in the
supernatant The electrophoretic heterogeneity of these
C-terminal fragments is compatible with their being
modi-fied by GPI anchors [26] The immunoprecipitated 19 kDa
protein was detectable only as a single 19-kDa band The
only strong [3H]GlcN-labelled band in the total extract
matching the Western blotted material was a 17-kDa band
The majority of the [3H]GlcN-labelled material ( 70% of
the total label) ran between 5 and 10 kDa and did not
comigrate with either Western blotted or
immunoprecipi-tated material It is likely that this fast-moving [3
H]GlcN-labelled material corresponds to the GPI-anchored peptides
no longer associated with MSP-1 C-terminal epitopes The
incorporated 3H-label in this 5–10 kDa material ran as
glucosamine by paper chromatography (data not shown),
indicating that [3H]GlcN had not been chemically
trans-formed With both antibodies, immunoprecipitation of
intact MSP-1195 kDa was more efficient than that of the
fragments On the contrary, Western blotting detected the
fragments more efficiently This probably reflects
conform-ational differences between MSP-1 intact protein and its
fragments in solution and adsorbed onto nitrocellulose The
detectability of [3H]GlcN-labelled, immunoprecipitated
fragments is therefore likely to be suboptimal
LowMrC-terminal fragments are released
in membrane vesicles by parasitized erythrocytes
The 5- to 17-kDa [3H]GlcN-labelled material pelleted with
in vitro released membrane vesicles (Fig 3, unselected)
corresponding to the nanovesicles released from normal
erythrocytes following Ca++exposure [27] This high-speed
pellet of released vesicles contained labelled 5- to 10- and 17-kDa fragments, as well as a labelled 19-kDa fragment immunoprecipitable with the anti-(C-ter) mAb (Fig 2; IP anti C-ter) The [3H]GlcN-labelled MSP-1 fragments detectable in the released vesicles were predominantly of low Mr No intact MSP-1 protein and no other C- or N-terminal fragments were detected by immunoprecipitation
in the released vesicles Importantly, endogenous CD59 was detected by Western blotting in the released membrane vesicles immunoselected with solid-phase anti-MSP-1 C-ter mAb (Fig 3, WB anti-CD59) The parasite extract from the same culture (whole extract) contained the full spectrum of MSP-1 protein and fragments, including [3H]GlcN-labelled
86 and 40 kDa N-terminal fragments detected by a polyclonal antibody (immune serum) directed against MSP-1 The absence of intact MSP-1 in the vesicles strongly suggested that they were free of parasites (merozoites) and consisted only of membranes emanating from the parasi-tized erythrocyte Moreover, the coexistence of MSP-1
19 kDa and CD59 in nanovesicle membranes selected with anti-(C-ter) mAb further suggests that MSP-1 and CD59 proteins are released in the same membrane vesicles from
P falciparum-infected erythrocytes
Analysis of the non-GPI-anchored carbohydrate moieties of MSP-1
It is therefore possible that part of the remaining protein-bound, non GPI-anchored [3H]GlcN label could be incor-porated on the surface of the molecule This contention was further supported by the observation that the 86- and 40-kDa N-terminal fragments which cannot carry the GPI anchor were strongly labelled fragments compared to the C-terminal ones These results were confirmed by exo-galactosylation of the affinity-purified MSP-1 proteins transferred to nitrocellulose membranes The specifically immunoprecipitated 195-kDa MSP-1, and 56-kDa C-ter-minal and 86-kDa N-terC-ter-minal fragments (marked with asterisks in Fig 2) were exogalactosylated with3 H-UDP-Gal at levels significantly above control labelling of the non O-GlcNAc-modified BSA (Table 1) Sham immunopreci-pitations with uninfected erythrocyte lysates did not yield exogalactosylated material above the BSA control at 195, 86 and 56 kDa (data not shown) Specificity of the b-1,4-galactosyl transferase-mediated labelling was confirmed by removal of the incorporated label following treatment with b-galactosidase Further, the Jack Bean b-N-acetylglucosa-minidase, an enzyme that specifically cleaves O-GlcNAc residues in b-anomeric linkage [22], released 65% of the [3H]GlcN label from biosynthetically labelled proteins im-munoprecipitated with either 3B10 or 7B2 mAbs (compare with C-ter and N-ter immunoprecipitates of pellet, Fig 2) Prediction of potential O-glycosylation sites on MSP-1 The potential for O-GlcNAc modification of five known and verified MSP-1 sequences (Ghana [28]; Uganda-Palo Alto; Papua New Guinea MAD 20; Thai K1 and Wellcome isolates [23]) was evaluated using the DictyOGlyc 1.1 predictor [24] Fig 4 (left panel) shows that Thr at position
1278 and Ser at 1280 (single cross), and Ser at positions 1498 and 1506 (asterisk) in the MSP-1 sequences of Ghana,
Trang 6PnGMAD20 and Uganda isolates had the potential to be
modified by a-GlcNAc The allelomorphic sequences of the
Wellcome and Thai-K1 strains bear deletions at these
positions [23] and thus could not be evaluated One Ser
however (1353, single cross) had a potential close to the
threshold line in the Thai and Wellcome sequences All of
these sites are located within the C terminus and correspond
to the allelomorphic block 5–16 of the MSP-1 sequence [23]
but do not encompass the GPI-anchored 19-kDa fragment
In contrast to the C-terminal region, the N-terminal region
does not contain any predictable a-GlcNAc sites
Interestingly, screening for potential b-O-GlcNAc
modi-fication sites revealed a wider and different set of sites (Fig 4,
right panel) b-O-GlcNAc sites occured both in the
N-terminal, nonpolymorphic (nonallelomorphic) part of
MSP-1 (blocks 1–4), as well as in the polymorphic
(allelo-morphic) part of MSP-1 (blocks 5–16) In block 1–4, multiple
threonines between the aligned positions 80–135 were
detectable in the Uganda sequence (filled triangles) In the
other four sequences, one cluster of threonines between 75
and 80 and another cluster of serines (135–145) were also
predicted In blocks 5–16, serines at aligned positions 931 and
957 were positive in the Ghana, Png and Uganda sequences
(filled triangles) The next positively predicted O-GlcNAc
sites occured at the aligned positions 1271 (Ser), 1278 (Thr)
and 1283 (Ser) (filled circles) Position 1271 was positive in
the Thai and Wellcome sequences while position 1283 was
positive for the Ghana, Png and Uganda sequences Position
1278 was the only position of the alignment where both
a- and b-O-GlcNAc have been predicted in the Ghana, Png
and Uganda sequences The Thr1503 was predicted positive
for b (Ghana, Png and Uganda: filled circles, right panel),
while Ser1498 and 1506 were positive for a (double cross, left panel) Thr1693 in block 17 was found positive for b only in the Png sequence and suggests that the 19 kDa C-terminal fragment does not usually contain an O-GlcNAc site in MSP-1
The M25 Zaı¨re MSP-1 shows substantial labelling in its
N terminus and thus fits the b-O-GlcNAc predictions made
on the Ghana, Papua and Uganda sequences
Distribution of MSP-1 fragments in Triton X-100 resistant and soluble membranes of the parasitized erythrocyte
GPI-anchored membrane proteins favour the environment
of ordered lipids [29] and accumulate in the low density, detergent-resistant membranes recovered after equilibrium density centrifugation of Triton X-100 lysates of mamma-lian cells [20] Parasitized erythrocytes were isolated by sedimentation in Percoll, washed and hypotonically lysed to remove haemoglobin (Fig 1C) The resulting ghosts were extracted in Triton X-100 before equilibrium density centrifugation in sucrose In this gradient system (Fig 1C and Fig 5), the Triton X-100 resistant membranes floated
to the 5–36% sucrose interface (fractions 3–4) while the membrane proteins that lack strong interactions with membrane lipids were solubilized (fractions 5–10) and the parasites were pelleted with remnants of the parasitopho-rous vesicular membrane (fraction 11) The floating mem-branes from such a Triton X-100 extract of parasitized erythrocytes were enriched in the 19-kDa C-terminal fragments of MSP-1 and in the erythrocytic, GPI-linked CD59 The Triton X-100 soluble fractions of the gradient contained the bulk of the proteins displayed in the gradient, but only small quantities of MSP-1 19 kDa and CD59, suggesting that the two GPI-linked proteins seek similar lipid-rich membrane environments
Discussion
MSP-1 not only ensures adhesion of newly released merozoites to fresh erythrocytes, but its C-terminal GPI-linked fragments also appear to redistribute in the parasi-tized erythrocyte in detergent-resistant membrane domains During the initial phase of this process, vesicle-borne MSP-1 fragments [30] could come in contact with glycosyltrans-ferases present in the erythrocyte cytosol [31,32], or in intracellular membranes [33,34] The intracellular localiza-tion of the glycosyltransferases that catalyse O-GlcNAc addition remains undefined and the O-GlcNAc transferase activity has been found in membrane-free reticulocyte lysates [32], as well as membrane-associated Most O-GlcNAc-modified proteins are indeed cytoplasmic or nuclear [15], but are also found at the cell surface [35] This implies that GPI-linked MSP-1 fragments exposed to the lumenal side of intracellular membranes such as the tubo-vesicular network [13,36] could become O-GlcNAc-modi-fied similarly to the O-GlcNAc-modiO-GlcNAc-modi-fied proteins found at the cell su rface [35]
Our evidence for carbohydrate modifications of MSP-1 other than the GPI anchor is based on the following: (a) [3H]GlcN biosynthetic labelling occurs in both the C- and N-terminal fragments; (b) exogalactosylation of terminal
Table 1 Exogalactosylation of MSP-1 protein and fragments From an
SDS extract of parasitized erythrocytes (four 10-mL culture plates)
obtained following hypotonic lysis of Percoll-isolated erythrocytes
containing late trophozoites and schizonts (P in Fig 1), C- and
N-terminal MSP-1 fragments were immunoprecipitated with 3B10
(anti-C-ter) or 7B2 (anti-(N-ter)) mAbs coupled to Sepharose The
195-kDa whole protein, and the 56-195-kDa C-terminal and the 86 195-kDa
N-terminal fragments were identified by immunoblotting of an aliquot
of the immunoprecipitate run in parallel The corresponding 195-,
86-and 56-kDa proteins were subjected to exogalactosylation in duplicate.
The data presented are representative of three different experiments.
Substrate
Galactosyl transferase
b-galacto-sidase c.p.m.
20 mU 10 mU 198
20 mU 10 mU 264
20 mU 10 mU 200
Trang 7O-GlcNAc also occurs in both termini of the MSP-1
molecule; and (c) 65% of the incorporated [3H]GlcN
associated with MSP-1 in an SDS parasite extract is
removed by Jack Bean glucosaminidase, an enzyme that
releases O-GlcNAc moieties in b-anomeric configuration
[22] The remaining 35% of incorporated [3H]GlcN that is
resistant to the Jack Bean hexosaminidase could be either
GPI-linked, N-linked to asparagines or linked to the
surface of the protein in a-O-GlcNAc configuration Two
a-O-GlcNAc sites are indeed predicted in the Ghana,
PngMAD20 and Uganda strain MSP-1 proteins and it is
remarkable that the three b-O-GlcNAc sites predicted in the
allelomorphic portion of MSP-1 (blocks 5–16) are distinct
from the predicted a-O-GlcNAc sites (with the exception of
Thr1278), and distinguish the two dimorphic forms (Ghana,
Png and Uganda vs Thai-K1 and Wellcome) Such
b-O-GlcNAc sites are not localized in the regions of
homology (387–413 and 1100–1187) within the sequences
of blocks 5–16 of the two dimorphic forms The comparison could not be extended to the a-O-GlcNAc sites because the MSP-1 proteins of Thai-K1 and Wellcome strains contain deletions [23] in the regions of MSP-1 where a-O-GlcNAc sites have been predicted
Our previous findings indicated that [3H]GlcN is linked to serines and [17,37,38] threonines [7,10] and we now show that O-GlcNAc addition takes place in both the C- and N-terminal ends of the protein, while the GPI-anchor remains the major carbohydrate modification of MSP-1, as established by others [17,37,38] However, following SDS/ PAGE separation of MSP-1 fragments, the labelled GPI anchors appear dissociated from the 19-kDa fragment carrying the C-terminal epitope (compare Figs 2 and 3) The lower Mr [3H]GlcN-labelled material amounts to
80% of the biosynthetically incorporated GlcNAc
Fig 4 a-GlcNAc and b-GlcNAc predictions on the aligned sequences of MSP-1 from Ghana (RO-33), Papua New Guinea (MAD20), Uganda (Palo Alto), Thailand (K1) and Wellcome strains Sequences extracted from SwissProt (accession no P19598, P08569, P50495, P04932, P04933) were aligned according to [23] and designated as G (Ghana), P (Png MAD20), U (Uganda), T (Thai) and W (Wellcome) Alpha- and b-GlcNAc site predictions, made using methods based on neural networks, were marked on the alignment The x-axis shows the position of the alignment, and the y-axis marks the predicted potentials The horizontal wavy line is a surface-accessibility derived threshold A vertical impulse crossing the threshold
is said to represent a (predicted) glycosylated site While the GlcNAc linkages in the N-terminal half of the protein are probably entirely the b form, the C-terminal half has a mix of a and b forms (Left) X marks represent potential a-GlcNAc positions For Ghana, PnG and Uganda strains, out
of the four potential positions 1278, 1280, 1498, 1506, 1278 may be a b-O-GlcNAc (Right) The triangles and circles represent N- and C-terminal predicted b-O-GlcNAc positions Empty circles and triangles are other possibles (negative predictions but very close to the threshold) The positions in the figure are alignment positions Exact sequence positions may vary slightly from strain to strain The prediction methods are available at http://www.cbs.dtu.dk/services/.
Trang 8(5–10, 17 and 19 kDa), but only the [3H]GlcN-labelled
19-kDa band is immunoprecipitated by the anti-(C-ter)
mAb The same mAb however, detects the 17-kDa fragment
and part of the lower Mrfragments by Western blotting, but
still does not recognize the bulk of the [3H]GlcN-labelled
peptides between 5 and 10 kDa Those [3H]GlcN-labelled,
lipophilic peptides are contained in sedimentable vesicular
membranes (nanovesicles, see [27]) released by parasitized
erythrocytes in culture This lipophilic behaviour strongly
suggest that MSP-1 C-terminal peptides carry [3
H]GlcN-labelled GPI anchors Vesicular membranes enriched in
GPI-anchored peptides could be a vehicle for the bioactive
inositol glycan moieties released by P falciparum parasites
[39] As the tyrosine kinase activity of macrophages was
shown to respond to the hydrophilic, carbohydrate moieties
of GPI molecules, released vesicles should exert their
biological effect by simple contact, whereas protein kinase
C enzymes were modulated only by acylated inositol glycans
and would thus require fusion of the vesicles with the target
cell membrane [39]
The low Mr C-terminal MSP-1 fragments, like the
endogenous GPI-linked CD59, are selectively enriched in
low density, detergent-resistant membranes of the
para-sitized erythrocytes, suggesting that the parasite
GPI-anchored proteins seek a similar environment as the
endogenous ones in the membranes of the parasitized
erythrocyte The presence of CD59 in nanovesicles
immu-noselected with anti-(C-ter) mAb strongly suggests that
both GPI-linked proteins are inserted in membrane subdo-mains of similar properties that vesiculate as a unit in form
of nanovesicles
The Triton X-100 resistant membranes described in this study are most probably derived from erythrocytes and not from the parasite membranes, as intact parasites were removed by centrifugation prior to sucrose gradient float-ation Sphingomyelin is synthesized de novo in the parasi-tized erythrocyte under the control of P falciparum [40] and may also contribute to the formation of detergent-resistant membrane domains in the newly made membranes The Triton X-100 resistant membranes originally described in erythrocyte ghosts were characteristically rich in sphingo-lipids and cytoskeletal proteins spectrin, actin and band 4.1 [41] Using standard equilibrium sucrose density gradients, Civenni et al have shown that GPI-linked surface proteins such as acetylcholinesterase, CD55 and CD59 are also included in the detergent-resistant membranes of normal erythrocytes [42] and released as vesicles by the stressed or aging erythrocytes [43] The considerable remodelling of the cytoskeleton–membrane interface taking place in the para-sitized erythrocyte [44] makes it difficult to define precisely the relationship of the GPI-rich, detergent-resistant mem-branes we describe with the detergent-resistant memmem-branes
of normal erythrocytes However, a recent study proposes that vacuolar uptake of erythrocyte components (CD59, Duffy antigen) could be carried out by membranes with detergent-resistant properties in the parasitized erythrocyte [19] We further show in this study that membranes containing GPI-linked MSP-1 C-terminal 19-kDa fragment and endogenous CD59 are released in vesicular form by the cultured parasitized erythrocyte The in vivo implication of this finding is that MSP-1 C-terminal antigens may disperse
in the bloodstream and possibly integrate other cellular membranes [45]
Acknowledgements This work was supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Grant ID
970604 and Swiss National Science Foundation Grant 31–57696.99.
R G thanks J Hansen for useful discussions, and the Danish National Research Foundation for funding We are grateful to Drs J A Lyon and V Horejsi for their kind gifts of antibodies.
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