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INTRODUCTION |
Glycosylphosphatidylinositol
(GPI)1 anchors represent a
special class of glycolipids consisting of a conserved
trimannosylglucosaminyl moiety linked to the inositol residue of PI.
GPIs are ubiquitous in eukaryotes (1, 2), and their primary function is
to anchor proteins to the cell membranes (1-3). Examples include the
Trypanosoma brucei variant surface protein, T. cruzi
trans-sialidase, Plasmodium falciparum MSP-1 (merozoite
surface protein 1) and MSP-2, Leishmania gp63, Thy-1,
acetylcholine esterase, CD14, CD52, CD55, and CD59 (3-7). In
parasites, GPIs also occur abundantly as free lipids (not anchored to
proteins). GPIs exhibit a wide array of functions (8, 9). GPIs from
different species differ in the type of acyl/alkyl substituents and the
presence of additional sugar moieties on third and/or first mannose and
ethanolamine phosphate residues on the carbohydrate moiety,
leading to a broad structural diversity that may relate to their
diverse biological activity (3).
GPIs of P. falciparum have been shown to induce secretion of
proinflammatory cytokines, including TNF-
and up-regulation of cell adhesion molecules (10-12). Because P. falciparum-infected erythrocytes sequester in the microvascular
capillaries of vital organs, the above process is expected to
synergistically promote adherence of parasite-infected erythrocytes;
this in turn potentiate proinflammatory immune responses in deep
endothelium because of local high concentration of GPIs and other
factors released by accumulated parasites, leading to organ dysfunction
and life threatening pathological conditions. Thus, GPIs of P. falciparum have been identified as "pathogenicity factors"
(13).
P. falciparum GPIs activate PTK and PKC pathways, which
collaboratively regulate the NF-
B/rel
transcription factors with the downstream expression of proinflammatory
immune responses (10-12). The conserved glycan core of GPIs,
Man
1-2Man
1-6Man
1-4GlcN
1-6myo-inositol, was
reported to be the minimum structure involved in PTK activation, whereas the diacylglycerol moiety has been postulated to be an independent second messenger for the PKC activation (12). Although it
has been predicted that a transmembrane receptor with a lectin-like property might be involved in GPI-mediated cell signaling (12), the
nature of the receptor and the sugar(s) involved in the receptor recognition has not been elucidated. The suggested involvement of
membrane-bound GPI-phospholipase D in hydrolyzing GPI and thus allowing
the DAG moiety to enter cells or GPI translocation across cell membrane
by the action of a "flippase" to activate PKC remains speculative
(12). Although it has been reported that the DAG moiety but not
a 1-alkyl-2-acylglycerol residue could activate PKC (12),
T. cruzi GPIs containing a 1-alkyl-2-acylglycerol moiety
exhibit potent cytokine-inducing ability, and
-Gal residues of these
GPIs were reported to be crucial for the activity (14, 15). These
observations suggest the involvement of multiple recognition mechanisms
in signal transduction by GPIs.
Recently we established, by mass spectrometry, the structures of GPIs
purified from intraerythrocytic P. falciparum (16). Here, we
studied the specific structural elements and nature of interaction
involved and the mechanism of cell signaling in GPI-induced TNF-
secretion. The data show that a novel interaction mediates cell
signaling and that the terminal fourth mannose and 1-acylglycerol residues are critical for GPI activity. Cell signaling is mediated by a
previously unrecognized mode of outside-in signal transduction without
inserting into cell membranes or internalization of GPIs.
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EXPERIMENTAL PROCEDURES |
Materials--
RPMI 1640, Dulbecco's modified Eagle's
culture medium, fetal bovine serum, and cell culture reagents were
purchased from Life Technologies, Inc. Human blood and serum
were from Interstate Blood Bank (Memphis, TN). Bee venom phospholipase
A2 (1800 units/mg), jack bean
-mannosidase (30 units/mg), soybean 1-palmitoyl-2-linoleoylphosphatidylinositol, bovine
liver 1-stearoyl-2-arachidonylphosphatidylinositol, synthetic dipalmitoylglycerol and dioleoylglycerophosphate, trifluoroacetic acid,
-methyl mannoside,
-methyl glucoside,
-methyl galactoside,
-(1-2)-linked mannobiose, soluble yeast mannan, and saponin were from Sigma. [6-3H]Glucosamine (23 Ci/mmol) and
[3H]myristic acid (55 Ci/mmol) were from Amersham
Pharmacia Biotech. Genistein and GF 109203X were gifts from Dr.
Toshio Kitazawa.
P. falciparum Cell Culture--
Culturing of intraerythrocytic
P. falciparum (FCR-3 strain), synchronization, and
mycoplasma testing were performed as described (17-19).
Metabolic Labeling of GPIs--
Metabolic labeling of
parasites with [3H]GlcN (50 µCi/ml) and
[3H]myristic acid (50 µCi/ml) was performed as described
(17).
Isolation of GPIs from P. falciparum--
Purification of
parasites and isolation of GPIs were performed as described (16).
Briefly, cultures with 20-30% parasitemia were harvested at
mid-schizont stage, enriched to 70-80% by gelatin floatation (20),
and lysed with 0.015% saponin. The erythrocyte debris were removed,
and the parasites were washed, lyophilized, and stored at
80 °C
(16). The parasite GPIs were extracted with chloroform/methanol/water
(10:10:3, v/v/v) and partitioned between water and water-saturated
1-butanol as described (16).
Nitrous Acid Treatment--
The HPLC-purified GPIs (20 µg) were treated with HNO2 in the presence of sodium
taurodeoxycholate (21, 22). The released PI moieties were extracted
with water-saturated 1-butanol and washed with water. The aqueous phase
was desalted on Bio-Gel P-4.
Cleavage by HF--
HPLC-purified GPIs (10 µg) were treated
with 50% aqueous HF (21, 22), and the reaction mixture was diluted
with 20 volumes of ice cold water, lyophilized, and partitioned between
water and water-saturated 1-butanol. The glycan and diacylglycerol
moieties were purified by HPLC (16).
Deacylation of GPIs--
The HPLC-purified GPIs (2 µg) were
deacylated with methanol, 30% ammonia (1:1, v/v) (16), and the glycan
moiety was solubilized in water and dried in a Speed Vac.
Treatment with Mannosidase and Phospholipase
A2--
HPLC-purified GPIs (10 µg plus 500,000 cpm of
[3H]GlcN-labeled GPIs) were treated separately with jack
bean
-mannosidase and bee venom phospholipase A2 (16,
23). The products were purified by HPLC and analyzed by HPTLC.
Macrophage Cell Culture--
The J774A.1 murine macrophage
cell line (ATCC) was cultured as monolayers in DMEM with 10% FBS. For
subculturing and cytokine induction, cells were detached by scraping.
Induction of TNF-
in Macrophages by P. falciparum
GPIs--
Freshly detached macrophages suspended in DMEM, 10% FBS
were seeded in 96-well microtiter plates (2.5 × 106
cells/well) and incubated at 37 °C. After 2 h, the nonadhered cells were removed, and cells were incubated with 200 µl of DMEM containing 10% FBS and varying amounts of GPIs, and other compounds added from stock solution in 80% ethanol, water, or
Me2SO. Cells treated with organic solvents (0.1%
final concentration) only were used as controls. For inhibition
studies, the compounds were added 1 h prior to the addition of
GPIs. The amount of TNF-
secreted was measured using a sandwich
ELISA kit (R & D Systems).
Uptake of GPIs by Macrophages--
Macrophages in microtiter
plates (2.5 × 106cells/well) were incubated with
[3H]GlcN- or [3H]myristic acid-labeled GPIs
(100,000 cpm each) in DMEM, 10% FBS for 6, 24, or 48 h. The
culture supernatants were collected, and aliquots were counted for
radioactivity by liquid scintillation counting. The cells were detached
by trypsinization, centrifuged, and washed with DMEM. The combined
washings and cells solubilized in 1% Triton X-100, 1% SDS were
measured separately for radioactivity.
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RESULTS |
Preparation and Purification of Specific Cleavage Products of P. falciparum GPIs--
Many parasitic protozoa, including P. falciparum, synthesize GPIs severalfold in excess of the amount
required to anchor proteins to cell surfaces. These GPIs are likely to
play major roles in membrane properties and the modulation of host
immune responses. P. falciparum GPIs have been shown to
elicit inflammatory cytokines including
TNF-
(10-13, 16).
To determine the structural feature(s) of parasite GPIs necessary for
the induction of TNF-
and to identify the receptor on the host
cells, GPIs were subjected to specific enzymatic and chemical
degradations, and the products were purified and tested. The
HPLC-purified GPIs
(EtN-P-Man4-GlcN-acyl-IP-DAG, designated as Man4-GPI; retention time, 92 min) were treated with jack
bean
-mannosidase and bee venom phospholipase A2 to
obtain GPIs lacking the distal fourth mannose residue
(EtN-P-Man3-GlcN-acyl-IP-DAG, Man3-GPI; HPLC retention time 92 min) and sn-2
fatty acid (EtN-P-Man4-GlcN-acyl-IP-MAG, sn-2 lyso-GPI; HPLC retention time 70 min),
respectively (Fig. 1). Both enzymes converted >95% GPIs into
their respective products (Fig. 1). The
acylated-inositol carbohydrate moiety with and without the phosphate
group on inositol (Man4-GlcN-acyl-IP and
Man4-GlcN-acyl-Ino) and the carbohydrate moiety
(OCH-CH2-P-Man4-AHM) that lacked the PI residue of GPIs were prepared by HF cleavage and nitrous acid deamination, respectively (Fig. 1). HPTLC indicated that these were
>95% pure (Fig. 2 and not shown). The
compound that lacked all three acyl substituents
(EtN-P-Man4-GlcN-IP-G) was prepared by
hydrolysis with ammonia in aqueous methanol. The identity of the above
products was established by determining mannose content, by identifying
the glycan core by specific degradative cleavage, and by analysis of
products by HPTLC and mass spectrometry (16, 17, 24).

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Fig. 1.
Schemes showing the enzymatic and chemical
cleavage sites for the preparation of GPI components. Treatment of
GPIs with -mannosidase gives
EtN-P-Man3-GlcN-acyl-IP-DAG. Treatment
with phospholipase A2 gives
EtN-P-Man4-GlcN-acyl-IP-MAG; HF gives
Man4-GlcN-acyl-IP, Man4-GlcN-acyl-Ino, and DAG;
and nitrous acid forms
OHC-CH2-P-Man4-AHM and
acyl-IP-DAG.
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Fig. 2.
HPTLC analysis of P. falciparum
GPI cleavage products. Analysis of the HPLC-purified
cleavage products of GPIs was carried out using Silica Gel 60 HPTLC
plates (Whatman) using chloroform/methanol/water (10:10:2.5, v/v/v).
After development, the plates were air-dried, sprayed with
En3Hancer (PerkinElmer Life Sciences), and exposed to x-ray
film at 80 °C. Lane 1, HPLC-purified GPIs; lane
2, mannosidase-treated GPIs; lane 3,
PLA2-treated GPIs; lane 4, HF-released
carbohydrate moiety extracted with water-saturated 1-butanol
(Man4-GlcN 1-acylIno);lane 5, HF-released
carbohydrate moiety recovered in aqueous phase
(Man4-GlcN 1-acylIno-P).
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Induction of TNF-
Secretion in Macrophages by P. falciparum
GPIs--
P. falciparum GPIs are known to elicit
proinflammatory cytokine responses by the activation of PTK and PKC
pathways by the carbohydrate and lipid moiety, respectively; these
signals cooperatively activate NF-kB/rel to
elicit cytokine responses (12). Although it has been predicted
previously that a lectin-type receptor is involved in the PTK
activation by GPIs, the nature of the sugar residue and the receptor
were not identified, and the mechanism of GPI interaction with
macrophages was not established. In this study, we tested purified GPIs
and various cleavage products of GPIs for the induction of TNF-
using murine macrophages. As reported previously (12, 16),
Man4-GPI was able to elicit TNF-
secretion by
macrophages (Fig. 3A).
However, in contrast to the previous report (12), Man3-GPI
was completely inactive, suggesting that the recognition of the
terminal mannose residue by the macrophage is crucial for the biologic
activity of GPIs (Fig. 3A). In agreement with this
observation,
-methyl mannoside,
-(1-2)-linked mannobiose, and
soluble yeast mannan inhibited the ability of P. falciparum GPIs to induce TNF-
secretion by >95%, whereas
-methyl
glucoside and
-methyl galactoside were not inhibitory (Fig.
3B). To determine whether the carbohydrate moiety of the
parasite GPIs alone is sufficient for the production of TNF-
,
macrophages were treated with HF-released carbohydrate moieties,
Man4-GlcN-acyl-IP and Man4-GlcN-acyl-Ino, and
with deacylated GPI,
EtN-P-Man4-GlcN-IP-G. All
three products failed to elicit TNF-
secretion (Fig. 3A). In addition, they effectively inhibited the activity of GPIs (Fig. 3B). The parasite GPI precursor lipids extracted with
chloroform/methanol (2:1, v/v), bovine liver and soybean PIs,
dipalmitoylglycerol, and dioleoylglycerophosphate were also unable
to elicit TNF-
secretion (Fig. 3B and not shown).
Together, these results suggested the critical involvement of the
distal fourth mannose and the requirement of intact GPIs for the
activity (12). Furthermore, in agreement with the previously reported
involvement of PTK and PKC activation by P. falciparum GPIs
in eliciting TNF-
secretion, genistein and GF 109203X, the
inhibitors of the respective kinases (10-12), blocked >90% of GPI
activity (Fig. 3B).

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Fig. 3.
Measurement of TNF-
secreted by macrophages in response to P. falciparum
GPIs. Murine macrophages were treated with indicated amounts
of GPIs or cleavage products for 48 h, and then culture
supernatants were assayed for TNF- as described under
"Experimental Procedure." For inhibition studies (panel
B), macrophages were incubated with the desired compound for
1 h before the addition of GPIs. The plotted values are averages
of two independent experiments; in each case TNF- was assayed in
duplicates. A, at each concentration, the bars
from left to right, respectively, represent
intact GPIs, sn-2 lyso-GPIs,
Man3-GPI, HF-released carbohydrate moieties, and
HNO2-released PI moiety. B, macrophages were
incubated with various inhibitors 1 h prior to treatment with 300 ng of intact GPIs/well. Control, no inhibitor;
Man3-GPI, 300 ng of mannosidase-treated GPIs;
HNO2-PI, 300 ng of the nitrous acid-released PI
moiety; Me Man, 350 ng of methyl mannoside;
Mannobiose, 600 ng; HF-Glycan, 300 ng of
HF-released carbohydrate moiety; Me Glc, 350 ng of methyl
glucoside; Me Gal, 350 ng of methyl galactoside;
Genistein, 5 µM; GF 109203X, 500 nM; Bovine PI, 300 ng of bovine liver PI.
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Because the lipid moiety of GPIs is also critically required for GPI
activity (see above), we examined whether the HNO2-released PI moiety, the parasite lipids extracted with chloroform/methanol (2:1), bovine liver and soybean PIs, and diacylglycerols could block
the activity of GPIs. Interestingly, none of these compounds could
inhibit the GPI activity (Fig. 3B). Furthermore, prior
incubation of macrophages with Man3-GPI had no effect on
the ability of Man4-GPI to elicit TNF-
production (Fig.
3B). These data suggest that initial recognition of the
distal fourth mannose residue is critical for the interaction of the
lipid moiety to activate the PKC pathway for productive cell signaling.
Thus, the lipid moiety-induced activation of PKC (12) must be tightly
regulated and coupled to PTK activation by the recognition of the
distal fourth mannose.
In a previous study, sn-2 lyso-GPIs have been
reported to be inactive (12). However, in this study, structurally
characterized sn-2 lyso-GPIs obtained by
phospholipase A2-treated Man4-GPI were able to
induce TNF-
secretion in a dose-dependent manner to
levels similar to those of Man4-GPI (Fig.
3A). Thus, our data demonstrate that the sn-2
fatty acyl residue is not required and 1-monoacylglycerol moiety of
P. falciparum GPIs is sufficient for TNF-
induction.
Localization of GPIs in Macrophage Cultures--
To
determine whether recognition of P. falciparum GPIs by
macrophages involves a mannose-specific receptor binding and/or uptake
of GPIs, macrophages were incubated with radiolabeled GPIs, HF-released
carbohydrate moiety, or deacylated GPIs. The distribution of these
compounds between culture medium and cells was analyzed. About 98% of
the radioactivity in intact GPIs was in the culture supernatants, and
about 1-2% was associated with the cells. This could be due to
nonspecific spontaneous insertion of GPIs into cell membranes, a known
phenomenon (25, 26). The HF-released carbohydrate moieties and
deacylated GPIs remained exclusively in the culture medium. These
results suggest that although the distal fourth mannose residue is
critical for the activity, GPI is not bound or internalized by
macrophages. In contrast, the macrophage mannose receptor binds and
internalizes compounds containing terminal mannose residues (27, 28).
These data taken together suggest that TNF-
production by P. falciparum GPIs is by unique interactions involving initial
recognition of the distal fourth mannose residue by a mannose-specific
component presumably localized in plasma membrane in association with
PTK.
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DISCUSSION |
In this study, highly purified GPIs from intraerythrocytic stage
P. falciparum and structurally characterized components of GPIs were assessed for TNF-
induction in macrophages to define the
active elements and the nature of receptor recognition. The data
presented here show that both the carbohydrate and lipid moieties of
GPIs are required for the activity. This is in agreement with previous
findings that the induction of proinflammatory cytokine responses by
P. falciparum and T. cruzi trypomastigote GPIs
involve a dual requirement for the glycan and diacylglycerol moieties; the GPI glycan moieties alone could not elicit TNF-
secretion (12,
15). Our study also identifies the critical requirement of the distal
fourth mannose residue for GPI activity. Furthermore, contrary to the
previous report (12), the results described here show that
sn-2 lyso-GPIs obtained by phospholipase
A2 treatment of the parasite GPIs are able to induce
TNF-
production to a level comparable with that induced by
intact GPIs.
The distal fourth mannose is a necessary component of P. falciparum GPIs for signaling TNF-
production. The following
data support this conclusion. 1) The removal of the distal fourth
mannose residue completely abrogates the ability of the parasite GPIs to induce TNF-
secretion in macrophages. 2)
-Methyl mannoside,
-(1-2)-linked mannobiose, mannan, the carbohydrate moiety released from GPIs by HF, and deacylated GPIs all could effectively inhibit the
ability of P. falciparum GPIs to elicit TNF-
secretion.
3)
-Methyl glucoside and
-methyl galactoside were completely
noninhibitory. Together, these data establish that induction of TNF-
secretion by P. falciparum GPIs occurs by the recognition of
the distal fourth mannose. This finding is in contrast to a previous
report that P. falciparum Man3-GPI was also
bioactive (12). The reason for the discrepancy is not clear. However,
it is possible that the reported activity of Man3-GPI could
be due to the presence of a significant amount of
Man4-GPI association in Man3-GPI fraction.
The requirement of the distal fourth mannose residue for the
P. falciparum GPIs to induce TNF-
secretion agrees with
the previously reported cytokine-inducing activity of T. cruzi trypomastigote mucin GPIs, which also contain unsubstituted
fourth mannose (14). The GPIs of T. cruzi trypomastigote
mucin have been shown to be severalfold more active than the GPIs of
P. falciparum (15). A comparison of the structures shows
that, unlike P. falciparum GPIs, the GPIs of T. cruzi trypomastigote mucin contain distal fourth
-mannose as
well as
-galactose residues linked to the first mannose. Therefore,
it appears that the presence of
-galactose residues further
potentiates interaction of T. cruzi GPIs for effective cell signaling.
Leishmania mexicana glycoinositolphospholipids containing a
1-alkyl-2-acylglycerol moiety and the conserved glycan core with
-(1-3)mannose linked to the first mannose residue but lacking ethanolamine phosphate on the third mannose could not induce
TNF-
production; however, these compounds could activate PTK (12). By comparing these results with those from the GPIs of P. falciparum and T. brucei variant surface protein, it
has been suggested that GPIs containing diacylglycerol but not those
with 1-alkyl-2-acylglycerol can elicit cytokine production (12).
However, because GPIs from T. cruzi trypomastigote mucin
with a 1-alky-2-acylglycerol residue can efficiently induce
TNF-
production (15), the presence of 1,2-diacylglycerol as opposed
to 1-alkyl-2-acylglycerol moiety is not a crucial requirement for GPI activity.
Not all GPIs containing terminal fourth mannose residue and
1,2-diacylglycerol or 1-alkyl-2-acylglycerol moiety can elicit TNF-
secretion in macrophages. As shown previously by Almeida et
al. (15), the GPI fraction of T. cruzi epimastigote
mucin that contains unsubstituted fourth mannose, as in the case of active GPI fraction of trypomastigote mucin and P. falciparum GPIs, is completely inactive. Whereas P. falciparum GPIs and the active GPI fraction of T. cruzi
trypomastigote both contain unsaturated acyl substituent at the
sn-2 position (15, 16), the inactive GPI fraction of the
T. cruzi trypomastigote mucin contains a saturated acyl
substituent at sn-2; the inactive GPIs of epimastigote mucin have a ceramide moiety. Therefore, as in the case of the active trypomastigote mucin GPIs (15), the unsaturated acyl group at the
sn-2 position seems to be required for the activity of
intact P. falciparum GPIs; however, P. falciparum
GPIs lacking sn-2 acyl substituent are also fully active
(see below).
It has been reported previously that sn-2
lyso-GPIs, obtained by the treatment of P. falciparum GPIs with phospholipase A2, were unable to
induce cytokine secretion (12). However, in our study, the
HPLC-purified structurally characterized sn-2
lyso-GPIs obtained from different GPI preparations were able
to reproducibly elicit TNF-
at levels similar to those elicited
by intact GPIs. The reason for this discrepancy is not known.
However, our data suggest that P. falciparum GPIs lacking
sn-2 acyl residue provide the necessary structural
requirements for cell surface interactions.
The mechanism of GPI-cell interaction in signaling is poorly
understood. Previous studies have predicted a receptor-mediated GPI
binding and insertion or internalization (12, 15). Interestingly, in
the present study, the P. falciparum GPIs were
neither bound to plasma membranes nor internalized by macrophages.
However, our data clearly indicate the critical participation of the
terminal mannose residue. Although the signal transduction by the well known macrophage mannose receptor to elicit cytokine responses involves
phagocytic or endocytic mechanism (29-32), our data suggest that
neither of these processes was involved in the case of P. falciparum GPIs. Furthermore, because neither the PI moiety
released by HNO2 nor Man3-GPI is able to
inhibit the activity of Man4-GPI, the interaction of the
acylglycerol moiety for PKC activation requires prior recognition of
the terminal mannose residue of GPIs. Moreover, because GPIs were not
inserted to cell membrane or internalized, the interaction of the GPI
acyl moiety in cell signaling represents a novel mechanism that is
likely to be tightly coupled to PTK activation. Further understanding
of the details of these interactions might offer specific targets for
malaria therapy.