From the Department of Biochemistry, Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, Scotland and the § Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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The major glycosylphosphatidylinositols (GPIs) in
African trypanosomes are glycolipid A, the precursor of the variant
surface glycoprotein membrane anchor, and glycolipid C, a species
identical to glycolipid A except that it contains an acylated inositol. Both glycolipids A and C contain dimyristoyl glycerol and are efficiently labeled with [3H]myristate in a
cell-free system. We now report a novel GPI known as lipid X. This GPI
is radiolabeled strongly with [3H]palmitate (and very
poorly with [3H]myristate or [3H]stearate)
in digitonin-permeabilized cells. The structure of lipid X is
Man1GlcNAc-(2O-palmitoyl)-D-myo-inositol-1-HPO4-3(lyso-palmitoylglycerol). Metabolically, lipid X exists as an intermediate, and can be detected only under conditions in which its formation is stimulated
(e.g. by EDTA) or its breakdown is inhibited
(e.g. by Co2+). Lipid X has not been observed
previously because these conditions do not support GPI biosynthesis. We
speculate that lipid X is an intermediate in the catabolism of
conventional trypanosome GPIs, possibly deriving from breakdown of
glycolipid C.
Most eukaryotes possess cell surface proteins that are anchored to
the plasma membrane by a covalently attached
glycosylphosphatidylinositol (GPI)1 (1-5). The conserved
core structure of a GPI is EtN-P-Man3-GlcN-PI, with the
ethanolamine residue being joined via an amide linkage to the
The trypanosomes are protozoan parasites that cause sleeping sickness
in humans and a related disease in cattle. These parasites live
extracellularly in their host's bloodstream and they evade the immune
response by a process known as antigenic variation. The variant antigen
is the GPI-anchored variant surface glycoprotein (VSG), which forms a
densely packed coat covering the entire surface of the parasite cell
(10). The GPI biosynthetic pathway in Trypanosoma brucei has
been elucidated using a cell-free system containing washed trypanosome
membranes (11-13). In this pathway, N-acetylglucosamine (GlcNAc) is first transferred from UDP-GlcNAc to phosphatidylinositol (PI), forming GlcNAc-PI via a sulfhydryl-dependent GlcNAc
transferase (14). This species is then de-N-acetylated to
form GlcN-PI (15-17). Subsequently, there is the addition of three
mannose residues (18), donated from dolichol phosphoryl-mannose (19),
to form Man3GlcN-PI. This intermediate is then
inositol-acylated (20) prior to the addition of ethanolamine-phosphate,
which is donated from phosphatidylethanolamine (21, 22), forming
glycolipid C'. After ethanolamine-phosphate addition there is a
subsequent inositol-deacylation, to form the GPI glycolipid A' (20).
This species then undergoes a series of fatty acid remodeling
reactions, in which the sn-1 and sn-2 fatty acids
are removed and replaced with myristate, forming the VSG precursor,
glycolipid A (12). Subsequently, there are myristate exchange reactions
which presumably serve as a proofreading mechanism to ensure that
myristate is the only fatty acid in the VSG GPI anchor (23, 24).
There are two free GPI species present in significant quantities in
T. brucei cells. One is glycolipid A, the VSG precursor, and
the other is glycolipid C, a species identical to glycolipid A except
that it has an extra fatty acid esterified to inositol (25-27). The
function of glycolipid C is not yet known, but glycolipids A and C are
in equilibrium via inositol acylation and deacylation reactions (20).
Surprisingly, trypanosomes synthesize much higher levels of GPIs than
they need for anchoring VSG (28, 29). There may exist a GPI catabolic
pathway which would prevent excessive accumulation of these molecules
(30). The experiments reported in this paper describe a novel T. brucei GPI, designated lipid X, which has some properties
suggesting that it is an intermediate in GPI breakdown.
Permeabilization of Trypanosomes with
Digitonin--
Trypanosomes (ILTat 1.3) from the buffy coat of
infected rat blood (27, 28) were washed twice (1,800 × g for 10 min at 4 °C) with permeabilization buffer (150 mM sucrose, 20 mM KCl, 3 mM
MgCl2, 20 mM Hepes-KOH, pH 7.9, 1 mM dithiothreitol, and 0.01 mg/ml leupeptin) and finally
resuspended at 6 × 108 cells/ml in permeabilization
buffer on ice. Digitonin (Sigma) was added to a final concentration of
0.03% (approximately 50 µg of digitonin/mg protein). This
concentration of digitonin had previously been shown to permeabilize
the plasma membrane of T. brucei leaving internal organelles
intact (31, 32). Following a 1-min incubation on ice, the cells were
centrifuged (12,000 × g for 5 min) and then washed
with 1.5 ml of permeabilization buffer. The cells were finally
resuspended at 6 × 108 cells/ml in permeabilization
buffer and stored in 0.5-ml aliquots at Preparation of the Trypanosome Cell-free System for GPI
Biosynthesis--
Lysates of trypanosomes were prepared and stored as
described previously (11), except that the incubation with tunicamycin prior to the hypotonic lysis was omitted.
Formation of
[3H]Palmitoyl-CoA--
[3H]Palmitic acid
(9,10-3H, 52 Ci/mmol, NEN Life Science Products Inc.) was
purified from contaminating fatty acids on a high performance thin
layer chromatography (TLC)-C18 reverse phase plate (Merck) eluted with
methanol/water (9:1, v:v). Approximately 1 mCi of fatty acid was
streaked along a line of 1 cm at the plate's origin. After elution,
the purified 3H-labeled fatty acids were extracted from
scrapings of the plate using methanol/pyridine/water (2:1:1, v:v) with
extensive vortexing and sonication. The organic extract was dried under
a stream of nitrogen and fatty acids partitioned by the addition of 0.5 ml of water-saturated butan-1-ol and 0.5 ml of butan-1-ol saturated water. After vortexing and centrifugation, the fatty acids were recovered in the upper butan-1-ol phase, which was dried in a Speed-Vac
evaporator. The purified fatty acids were resuspended at 1 mCi/ml in 80 mM Tris-HCl, pH 8.1, 120 mM KCl, 1 mM dithiothreitol, 5 mM ATP, 1 mM
CoA, 12 mM MgCl2, 0.1 mM EGTA, and
incubated with 1 unit/ml of Pseudomonas acyl-CoA synthetase
(Sigma) for 45 min at 37 °C. The reaction was terminated with
sufficient chloroform/methanol (2:1, v:v) to yield
chloroform/methanol/water (8:4:3, v:v), forming two phases. The lower
chloroform-rich phase was washed with an equal volume of aqueous upper
phase from a mock 8:4:3 partition. Purified 3H-labeled
palmitoyl-CoA, recovered in the combined aqueous phases, was stored at
Assay of Incorporation of [3H]Palmitic Acid into Lipids
in the Cell-free System and Permeabilized Trypanosomes--
Cell
membranes (1.5 × 107 cell equivalents) were suspended
in 25 µl of incubation buffer (110 mM sucrose, 10 mM Hepes-KOH, pH 7.9, 1 mM dithiothreitol, and
0.01 mg/ml leupeptin) which in some cases was supplemented with
CoCl2 or diisopropyl fluorophosphate (DFP). The suspension
was added to [3H]palmitoyl-CoA which had been dried in
the tube (0.2 µM final concentration) and the reaction
mixture was incubated at 37 °C. The reaction was terminated by the
addition of 0.25 ml of water-saturated butan-1-ol and 0.25 ml of
butan-1-ol saturated water. After vortexing and centrifugation, the
radiolabeled lipids were recovered in the upper butan-1-ol phase, which
was dried in a Speed-Vac evaporator and analyzed by TLC.
Enzymatic Digestions and Chemical Treatments of
Lipids--
GPI-PLD digests (29) were performed in 25 µl of 20 mM Tris-HCl, pH 7.4, 0.1 mM CaCl2,
0.008% Triton X-100 containing 0.2 unit of recombinant GPI-PLD
(Boehringer-Mannheim). PI-PLC digests (33) were performed in 50 µl of
20 mM Tris acetate, pH 7.4, 0.1% Triton X-100 containing 2 units of Bacillus thuringiensis PI-PLC (Oxford
GlycoSystems). Jack bean Formation of Lipid Standards for
TLC--
[3H]Myristate-labeled Man3GlcN-PI
standard was formed as previously reported (14, 16). The
[3H]myristate-labeled glycolipids A and C standards was
formed using the same procedure except the trypanosomes were labeled
with [3H]myristate in the absence of
phenylmethanesulfonyl fluoride (11).
[3H]Myristoyl-glycerol standard was formed from
[3H]myristate-labeled glycolipid A. This species was
digested for 2 h at 37 °C with 0.005 units of
Crotalus adamanteus phospholipase A2
in 30 µl of 25 mM Tris-HCl, pH 7.5, 2 mM
CaCl2, 0.1% sodium deoxycholate. Glycolipids were
recovered by extracting with water-saturated butan-1-ol as described
above. The butan-1-ol phase was dried in a Speed-Vac evaporator and the
glycolipids were then suspended in 30 µl of 50 mM
Tris-HCl, pH 8.0, 5 mM EDTA, 1% Nonidet P-40 and digested
with 0.15 unit of recombinant GPI-PLC (33) for 18 h at
37 °C. [3H]Myristoyl-glycerol was recovered by
extracting with water-saturated butan-1-ol as described above.
Synthetic
1-D-6-O-(2-amino-2-deoxy- Thin Layer Chromatography--
GPI samples were applied in 5 µl of chloroform/methanol/water (10:10:3, v/v) to Silica Gel 60 HPTLC
aluminum-backed plates (Merck). Plates were developed in solvents
system A, chloroform/methanol/water (10:10:3, v/v); B, chloroform,
methanol, 1 M acetic acid, water (25:15:4:2, v/v); C,
chloroform, methanol, 1 M ammonium acetate, 13 M ammonia/water (180:140:9:9:23, v/v). High performance
TLC-C18 reverse phase plates, developed in chloroform/methanol/water
(5:15:1, v:v), were used to resolve fatty acid methyl esters (35). TLC plates were analyzed by fluorography after spraying with
EN3HANCE.
Bulk Purification of Lipid X for Structural
Characterization--
Trypanosomes (1 × 1011 cells)
were permeabilized with 0.03% digitonin and resuspended in incubation
buffer. The suspension was incubated with
[3H]palmitoyl-CoA (0.2 µM) for 1 min at
37 °C followed by the addition of 10 µM cold
palmitoyl-CoA and a further incubation for 1 min at 37 °C. After the
incubation, the reaction was terminated by the addition of 50 ml of
water-saturated butan-1-ol and the lipids were recovered in the upper
butan-1-ol phase, which was dried in a rotary evaporator and
fractionated by TLC using solvent system A. The purified lipid X band
was further resolved by TLC using solvent system B. Subsequently lipid
X was resuspended in 20% propan-1-ol, 10 mM ammonium
acetate prior to applying to a reverse phase HPLC column (Kromasil 5 µm C8; 25 cm × 4.6 mm) equilibrated with the same solvent. The
column was eluted with a linear gradient to 90% propan-1-ol, 10 mM ammonium acetate over 80 min with a flow rate of 1 ml/min.
The highly purified lipid X was dried and resuspended in 20%
propan-1-ol, 1 mM ammonium acetate prior to applying to a
microbore reverse phase HPLC column (Kromasil 5 µm C8; 5 cm × 0.5 mm) equilibrated with the same solvent. This column was connected
online to a Micromass Quattro electrospray mass spectrometer to acquire
negative-ion mass spectra over the range m/z 600-1600. The
column was eluted with a linear gradient to 90% propan-1-ol, 1 mM ammonium acetate over 50 min with a flow rate of 10 µl/min. The electrospray source conditions were optimized using
synthetic GlcN-PI. Scans were averaged and processed using MassLynx software.
Radiolabeling of Lipid X with
[3H]Palmitoyl-CoA--
The initial indication of the
existence of lipid X came when we incubated trypanosomes permeabilized
with 0.03% digitonin with [3H]palmitoyl-CoA. TLC
analysis of the extracted radiolabeled lipids revealed a novel species,
designated lipid X, in addition to free fatty acids and phospholipids.
Lipid X had an RF slightly higher than that of a
glycolipid C standard (Fig. 1, lane
1) (glycolipid C is a free GPI with an acylated inositol
(25-27)). Lipid X was labeled in only trace amounts in the presence of
3 mM MgCl2 (Fig. 1, lane 2), but
production was greatly stimulated by chelation of the Mg2+
with EDTA (Fig. 1, lane 3). It was detected at low levels if Mg2+ was excluded from the reaction mixture (Fig. 1,
lane 4), but a substantial stimulation in formation of lipid
X was obtained in the presence of 1 mM CoCl2
(Fig. 1, lane 5). This stimulation with Co2+ was
almost completely reversed by the addition of EDTA (Fig. 1, lane
6). We obtained essentially similar results using washed trypanosome membranes obtained from osmotically lysed cells. Using standard cell-free system conditions previously optimized for GPI
biosynthesis (11) (containing 5 mM MgCl2, 5 mM MnCl2), we observed no labeling of lipid X
with [3H]palmitoyl-CoA (Fig. 1, lane 7).
However, when 3 mM MgCl2 provided the only
divalent cation, barely detectable amounts of lipid X were formed (Fig.
1, lane 8) which could be more easily visualized upon longer
exposures (data not shown). As with the digitonin-permeabilized cells,
production of lipid X was further enhanced by chelating the
MgCl2 with EDTA (Fig. 1, lane 9) or by excluding
it from the reaction mixture (Fig. 1, lane 10). As with the
permeabilized cells CoCl2 (1 mM) had an even
greater stimulatory effect (Fig. 1, lane 11).
In the following paragraphs we shall demonstrate that lipid X is a
novel GPI. Because there is little if any, of this species produced in
the presence of 5 mM MgCl2, 5 mM
MnCl2 (the standard conditions for GPI biosynthesis in the
cell-free system), it had not been detected in previous studies.
Surprisingly, lipid X is labeled to a 2-fold higher level in digitonin
permeabilized cells than in washed membranes. This finding contrasts
strikingly with the labeling of GPIs by radiolabeled UDP-GlcNAc and
GDP-Man; in the presence of 5 mM MgCl2, 5 mM MnCl2, GPIs are labeled approximately 20 times more efficiently in the cell-free system than in permeabilized cells (data not shown). Because of the more efficient labeling of lipid
X, we used permeabilized cells for the rest of the experiments in this paper.
Labeling of Lipid X with Different Fatty Acyl-CoAs--
It was
surprising that lipid X labeled strongly with
[3H]palmitoyl-CoA, as other trypanosome GPIs only label
efficiently with [3H]myristate, either by a fatty acid
remodeling (27) or by an exchange (23) pathway. We therefore tested
[3H]myristoyl-CoA, [3H]palmitoyl-CoA, and
[3H]stearoyl-CoA for their ability to label lipid X in
digitonin-permeabilized trypanosomes (the incubation buffer had no
added divalent cations). We found that lipid X was not labeled with
[3H]myristoyl-CoA or [3H]stearoyl-CoA (Fig.
2, lanes 1 and 5,
respectively) but that small amounts of this lipid were produced with
[3H]palmitoyl-CoA (Fig. 2, lane 3). The
labeling of lipid X by all three fatty acyl-CoA donors was enhanced
when the incubations were performed in the presence of 1 mM
CoCl2 (Fig. 2, lanes 2, 4, and 6),
but that labeling was greatest by far with
[3H]palmitoyl-CoA. Measurement of radioactivity in lipid
X (formed in the presence of 1 mM CoCl2) using
a TLC scanner indicated that labeling with
[3H]palmitoyl-CoA was at least 20-fold greater than with
[3H]myristoyl-CoA or [3H]stearoyl-CoA.
Lipid X Is a GPI--
To determine whether lipid X is a GPI, we
measured its sensitivity to GPI-specific phospholipase D (GPI-PLD). In
this experiment we first purified [3H]palmitate-labeled
lipid X on solvent system A, and then fractionated it again on system
B, an acidic system. The second fractionation revealed a
3H-labeled contaminant (indicated by an arrow)
that was sometimes present in preparations of lipid X (Fig.
3A, lane 3). GPI-PLD treatment
resulted in cleavage of lipid X to a product that comigrated with an
authentic lyso-phosphatidic acid standard (Fig. 3A,
lane 4). As a control, [3H]myristate-labeled
glycolipid A (Fig. 3A, lane 1) was also susceptible to
GPI-PLD digestion, forming dimyristoyl-phosphatidic acid which had a
TLC mobility close to that of a dipalmitoyl-phosphatidic acid standard
(Fig. 3A, lane 2). The fact that the only radiolabeled product of lipid X produced by GPI-PLD was lyso-phosphatidic
acid indicated that all of its [3H]palmitate is linked to
glycerol. We also analyzed the radiolabeled fatty acids on lipid X by
producing fatty acid-methyl esters and resolving them by reverse phase
TLC. We found that the radiolabeled fatty acids were exclusively
palmitate, with no detectable myristate (data not shown).
We also found that lipid X labeled with [3H]myristoyl-CoA
or [3H]stearoyl-CoA (see Fig. 2, lanes 2 and
6) is sensitive to GPI-PLD, forming a
lyso-phosphatidic acid (data not shown). Furthermore, the
latter two forms of lipid X contained either
[3H]myristate or [3H]stearate, with no
contaminating [3H]palmitic acid, as determined by
fatty acid-methyl ester analysis using reverse phase TLC (data not shown).
The susceptibility of lipid X to GPI-PLD indicated that it is a
previously unrecognized trypanosome GPI species. This fact, together
with its novel conditions for radiolabeling and its specific incorporation of [3H]palmitate, stimulated us to
investigate further both its structure and its metabolism.
Enzymatic and Chemical Characterization of Lipid X--
We used
enzymatic digestions and chemical treatments to characterize lipid X
that had been TLC purified using both solvent systems A and B (Fig.
3B, lane 1). The resistance of lipid X to PI-PLC (Fig.
3B, lane 2) (36), together with its susceptibility to
GPI-PLD (Fig. 3A, lane 4), suggests that lipid X has a fatty acylated inositol residue.
If lipid X lacks a phosphoethanolamine, then it should be sensitive to
JBAM, an enzyme which removes all mannose residues from a T. brucei-free GPI which lacks phosphoethanolamine (14, 37). As a
control for the JBAM treatment, authentic
[3H]myristate-labeled Man3GlcN-PI was
digested to form [3H]myristate-labeled GlcN-PI (Fig.
3B, lane 11). Approximately 70% of lipid X was susceptible
to JBAM digestion (as judged by densitometry of the autoradiograph),
forming a product (Fig. 3B, lane 3) that migrated close to
the GlcN-PI standard (Fig. 3B, lane 11). These results
suggest that lipid X lacks phosphoethanolamine and contains at least
one mannose residue. Lipid X comigrated with a
[3H]myristate-labeled Man1GlcNAc-PI standard
in other TLC solvent systems (data not shown).
Another characteristic of GPIs is sensitivity to cleavage by nitrous
acid (HONO) deamination (38). Surprisingly, lipid X proved to be
resistant to HONO treatment (Fig. 3B, lane 4). This finding
contrasted with that of glycolipid A or Man3GlcN-PI,
conventional GPIs which formed PI on treatment with HONO (Fig. 5,
lanes 8 and 10). The resistance of lipid X to
HONO suggests that it does not contain a free amino group on its
glucosamine residue. A possible explanation would be that it contained
N-acetylglucosamine. Taken together, all of these
experiments suggest that lipid X could have the structure:
Man1GlcNAc-(2-O-acyl)-myo-inositol-1-HPO4-lyso-palmitoylglycerol.
Structural Analysis of Lipid X Using Electrospray Mass
Spectrometry--
We purified [3H]palmitate-labeled
lipid X from 1011 digitonin-permeabilized trypanosomes by 2 consecutive preparative TLC steps (using solvent systems A and B)
followed by RP-HPLC on a C8 column eluted with a gradient of
propan-1-ol. Lipid X eluted as a single species at approximately 65%
propan-1-ol (just after the elution position of an authentic
[3H]myristate-labeled glycolipid A standard). An aliquot
of purified lipid X was applied to a microbore C8 RP-HPLC column and
eluted with a gradient of propan-1-ol. In this system, radioactive
lipid X eluted with virtually the same retention time as synthetic
(di-palmitoyl)GlcN-PI (Figs. 4,
A and B). Another aliquot of purified lipid X was
applied to the microbore column but this time with the eluate connected online to an electrospray mass spectrometer. Negative ion electrospray mass spectrometry spectra were collected over the range m/z
600-1600. At the elution position of radiolabeled lipid X, only one
major ion (at m/z 1175) was detected (Fig.
5) and the selected ion chromatogram of
this ion is shown in Fig. 4C. This ion is consistent with
the average mass of an [M Rapid Formation and Breakdown of Lipid X--
We studied the
kinetics of lipid X labeling. Labeling of lipid X with
[3H]palmitate in the presence of Co2+ occurs
very rapidly with significant levels detectable after 1 min incubation
(Fig. 6A, lane 1) and maximum
labeling occurring after 5-10 min (Fig. 6A, lanes 2 and
3). Thereafter, lipid X breakdown exceeds formation and
there is a slow decline of label in this species (Fig. 6A, lanes
4 and 5). In the absence of Co2+, maximum
labeling of lipid X is reached slightly earlier, after only 1 min
incubation, and there exists a subsequent more rapid breakdown (Fig.
6B, lanes 1-4; in this experiment there is a radiolabeled contaminant that migrates just below lipid X).
Further investigation revealed that lipid X breakdown could be
increased so that it greatly exceeded its formation. Lipid X was
labeled for 5 min in the presence of 3 mM MgCl2
and 10 mM EDTA (Fig. 6C, lane 1). Subsequently,
membranes were centrifuged to remove buffer containing Mg2+
and EDTA. After resuspending membranes in incubation buffer they were
incubated in the absence of divalent cations (Fig. 6C, lanes 2-5), in the presence of 3 mM MgCl2 (Fig.
6C, lanes 6-9) or in the presence of 1 mM
CoCl2 (Fig. 6C, lanes 10-13) for 1, 5, 15, and
30 min. Upon removal of EDTA, lipid X in the absence of divalent cations disappeared almost completely after only 1 min incubation at
37 °C (Fig. 6C, lane 2). There was a similar rate of
breakdown in the presence of 3 mM MgCl2 (Fig.
6C, lane 6). However, breakdown was reduced substantially
with the inclusion of 1 mM CoCl2 in the
reaction mixture, with significant levels of lipid X existing for at
least 5 min (Fig. 6C, lane 11) and with some remaining even
at 15 min (Fig. 6C, lane 12). All these results indicate that lipid X is an intermediate in a metabolic pathway, with
Mg2+ inhibiting its formation and Co2+
inhibiting its breakdown.
Possible Role of Lipid X in GPI Catabolism in Trypanosomes--
In
an attempt to study lipid X breakdown we found that DFP reduces the
steady-state level of this species. We incubated permeabilized trypanosomes with [3H]palmitoyl-CoA in the presence of 0, 0.1, 1, and 10 mM DFP. As shown in Fig.
7 (lanes 1-4), increasing the
concentration of DFP decreased the labeling of lipid X, with
concomitant labeling of comparable amounts of another species,
designated lipid Y. Lipid Y comigrated with an authentic
lyso-phosphatidic acid standard and the product of lipid X
digested with GPI-PLD (data not shown). We purified lipid Y (Fig.
7, lane 6), and digested it with alkaline phosphatase (Fig.
7, lane 7), yielding a lipid (marked by arrow) which migrated close to a [3H]myristoyl-glycerol standard
(Fig. 7, lane 5). Analysis of this species using reverse
phase-TLC provided further evidence that this species is
palmitoyl-glycerol (data not shown). Therefore we conclude that lipid Y
has the structure lyso-palmitoyl-phosphatidic acid. In
"Discussion" we will speculate on the possible relationship of
lipid X and lipid Y.
We have discovered a novel GPI in T. brucei which we
have designated lipid X. The structure of lipid X is
Man1GlcNAc-(2-O-palmitoyl)-myo-inositol-1-HPO4-lyso-palmitoylglycerol. We detected lipid X by incubation of digitonin-permeabilized
trypanosomes with [3H]palmitoyl-CoA, and we found that
its production was stimulated when Mg2+ and
Mn2+ (both required for GPI biosynthesis) were reduced in
concentration or removed (Fig. 1, lanes 2 and 3).
Production was stimulated even further on addition of 1 mM
Co2+ (Fig. 1, lane 5). We observed similar
effects of cations on the production of lipid X in washed trypanosome
membranes (which are highly efficient in supporting GPI biosynthesis in
the presence of 5 mM MgCl2, 5 mM
MnCl2), although the level of lipid X was 2-fold higher in
the permeabilized cells. Lipid X had not been observed previously
because ionic conditions which favor GPI biosynthesis greatly reduce
its labeling.
The kinetics of formation of lipid X are extremely rapid. For example,
there is maximal labeling of lipid X within 1 min (Fig. 6B, lane
1), and under other conditions it was degraded equally rapidly
(Fig. 6C, lanes 2 and 6). We were able to detect
large amounts of this species only by manipulation of the ionic
conditions. Evidence presented in Fig. 6 indicates that lipid X is a
transient species, with Mg2+ inhibiting its formation and
Co2+ inhibiting its breakdown. Such an intermediate could
be involved in GPI biosynthesis or breakdown. Since its structure does
not fit into the well known pathway of biosynthesis, we can speculate that it is an intermediate in GPI catabolism. It is already known that
GPIs are synthesized at a level about 10-fold greater than that needed
to provide GPI anchors for VSG, raising the possibility of a catabolic
pathway (30).
What could be the nature of the catabolic pathway? Glycolipid A (the
GPI precursor) is known to be in equilibrium with glycolipid C
(identical to glycolipid A, except for having an acylated inositol). The equilibrium is maintained by an inositol acylase and a deacylase (20). The function of glycolipid C is not known, but one possibility (already suggested in Ref. 30) is that it is an intermediate in GPI
catabolism. Lipid X could form from glycolipid C by removal of
phosphoethanolamine and two mannose residues, N-acetylation of the glucosamine residue, and subsequent removal of the two myristates from glycerol; the glycerol would then be acylated by a
single palmitate, forming lipid X. Lipid X could subsequently break
down and possible products could be
palmitoyl-lyso-phosphatidic acid (lipid Y in Fig. 7) and
another more polar species containing palmitoylinositol. According to
this scheme, DFP would allow the breakdown of lipid X but inhibit
further metabolism of lipid Y. An alternative pathway for catabolism
would involve a phosphatidic acid exchange mechanism (Fig.
8). In this scheme glycolipid C would be
degraded to a Man1GlcNAc-(acyl)PI and the resulting
Man1GlcNAc-(acyl)inositol headgroup would be exchanged with
a preformed palmitoyl-lyso-phosphatidic acid, forming lipid
X. The (dimyristoyl)phosphatidic acid moiety would be rapidly degraded
releasing myristate that could be recycled into the fatty acid
remodeling and exchange pathways. The lipid X would then be
cleaved into a Man1GlcNAc-(acyl)inositol headgroup for
further degradation and a palmitoyl-lyso-phosphatidic acid. This lyso-phosphatidic acid could then be recycled in the
phosphatidic acid exchange mechanism. The levels of lipid X and
palmitoyl-lyso-phosphatidic acid may be low but would
continually recycle. Only when we disturb the cycle with
Co2+ or DFP are we able to observe these intermediates.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-carboxyl group of the protein's C-terminal residue (6). Many
anchors have modifications, such as extra sugars or
ethanolamine-phosphate groups, linked to the conserved core (7).
GPI anchors contain either glycerolipids (diacylglycerol,
alkylacylglycerol, or lyso-acylglycerol) or ceramide lipid
moieties (7). Some anchors contain an extra fatty acid ester-linked to
the 2-hydroxyl of the inositol (8, 9).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
70 °C.
20 °C and dried into tubes as required.
-mannosidase (JBAM) digests were performed
in 30 µl of 0.1 M sodium acetate, pH 5.0, 0.1% sodium
taurodeoxycholate containing 0.5 unit of enzyme (Boehringer-Mannheim). All digests were performed for 18 h at 37 °C. Nitrous acid
(HONO) deaminations were performed in 50 µl of 50 mM
sodium acetate, pH 4.0, 500 mM NaNO2, and
0.01% Zwittergent 3-16 for 3 h at 60 °C (30). Alkaline
phosphatase digests were performed in 20 µl of 50 mM
Tris-HCl, pH 8.5, 0.1 mM EDTA, 0.1% NP-40 containing 2 units of calf intestinal alkaline phosphatase (Boehringer Mannheim) for
2 h at 37 °C. Lipids after all digestions and treatments were recovered by extracting with water-saturated butan-1-ol as described above.
-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-palmitoyl-sn-glycer-3-ylphosphate),
referred to herein as GlcN-PI, was synthesized according to
Cottaz et al. (34).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Formation of
[3H]palmitate-labeled lipids in digitonin-permeabilized
trypanosomes and in a cell-free system. Digitonin-permeabilized
trypanosomes (lanes 2-6) or membranes from osmotically
lysed trypanosomes (lanes 7-11) were incubated with
[3H]palmitoyl-CoA for 15 min at 37 °C. These
incubations were performed in the presence of 5 mM
MgCl2 and 5 mM MnCl2 (lane
7), 3 mM MgCl2 (lanes 2 and
8), 3 mM MgCl2 and 10 mM
EDTA (lanes 3 and 9), no additions (lanes
4 and 10), 1 mM CoCl2
(lanes 5 and 11), or 1 mM
CoCl2 and 2 mM EDTA (lane 6).
[3H]Myristate-labeled glycolipid C standard is shown
(lane 1). Radiolabeled lipids were extracted and analyzed by
TLC using solvent system A and fluorography. O, origin;
F, solvent front; FA-CoA, palmitoyl-CoA;
PL, phospholipids; FA, fatty acids;
NL, neutral lipids; FA-G, palmitoyl-glycerol;
X, lipid X. A concentration of 0.03% digitonin was used to
permeabilize the trypanosomes as it produced optimal levels of lipid X
formation. Production of lipid X was reduced by approximately 95%
using cells permeabilized with digitonin below 0.01% or above 0.04%.
Lanes 1-3 and 7-9 were from one experiment
(fluorograph developed after 72 h), and lanes 4-6 and
10 and 11 were from a second similar experiment
(fluorograph developed after 24 h). In some experiments
(e.g. lanes 2 and 3) we separated
palmitoyl-glycerol from palmitate whereas in others (e.g.
lanes 4-6) only one band was observed.
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Fig. 2.
Fatty acid specificity of lipid X
formation. Digitonin-permeabilized trypanosomes were incubated
with [3H]myristoyl-CoA (lanes 1 and
2), [3H]palmitoyl-CoA (lanes 3 and
4), or [3H]stearoyl-CoA (lanes 5 and 6) in the presence (lanes 2, 4, and
6) or absence (lanes 1, 3, and 5) of 1 mM CoCl2 for 15 min at 37 °C. Radiolabeled
lipids were extracted and analyzed by TLC using solvent system A and
fluorography. The slight difference in TLC mobility of lipid X
(X) formed by the various 3H-labeled fatty
acyl-CoA donors is due to differences in hydrophobicity of the
incorporated fatty acids. O, origin; F, solvent
front.
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Fig. 3.
Enzymatic and chemical characterization of
lipid X. A, lipid X purified by solvent system A was
incubated in the absence or presence of GPI-PLD and resolved by TLC
using solvent system B (lanes 3 and 4). A
contaminant in the lipid X preparation is indicated by the
arrow. [3H]Myristate-labeled glycolipid A
(Gly A) standard was also incubated in the absence or
presence of GPI-PLD (lanes 1 and 2). Synthetic
standards (PA,
1,2-dihexadecanoyl-sn-glycero-3-phosphate;
lyso-PA, oleoyl-sn-glycero-3-phosphate) were
detected by iodine staining. O, origin; F,
solvent front. B, lipid X was TLC purified using solvent
systems A and B. Subsequently lipid X (lane 1) was treated
with PI-PLC, JBAM, and HONO (lanes 2-4, respectively).
[3H]Myristate-labeled glycolipid A standard (lane
5) was treated with PI-PLC, JBAM, and HONO (lanes 6-8,
respectively). [3H]Myristate-labeled
Man3GlcN-PI standard (lane 9) was treated
with HONO and JBAM (lanes 10 and 11). The lipids
were then resolved by TLC using solvent system C. The altered TLC
mobility of lipid X after HONO treatment was caused by the Zwittergent
3-16 detergent. O, origin; F, solvent
front.
H]
ion of a molecule
with the composition: Hex1, HexNAc1,
inositol1, (HPO4)1,
glycerol1, palmitate2. These data are
consistent with the other analyses and suggest that the acyl chain
attached to the inositol ring is also palmitic acid. The proposed
structure of lipid X may therefore be refined to
Man1GlcNAc-(2-O-palmitoyl)-myo-inositol-1-HPO4-lyso-palmitoylglycerol. To evaluate the biological significance of this molecule, our next
studies, described below, concerned the metabolism of lipid X.
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Fig. 4.
HPLC and LC-MS of lipid X. A,
the elution of synthetic
D-GlcN 1-6-D-myo-inositol-1-HPO4-(sn-1,2-di-palmitoylglycerol)
(GlcN-PI) from the microbore RP-HPLC column. Selected ion
chromatogram of the [M
H]
pseudomolecular ion
of GlcN-PI at m/z 970. B, the elution of
3H-labeled lipid X from the microbore RP-HPLC column.
C, selected ion chromatogram of m/z 1175, the
major ion that coelutes with 3H-labeled lipid X.
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Fig. 5.
Negative ion electrospray mass spectra of
lipid X. Mass spectrum (m/z 600-1600) of the region of
the total ion chromatogram that co-eluted with 3H-labeled
lipid X (see Fig. 4B).
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Fig. 6.
Metabolism of lipid X. A,
digitonin permeabilized trypanosomes were incubated with
[3H]palmitoyl-CoA in the presence of 1 mM
CoCl2 for 1, 5, 10, 15, and 30 min at 37 °C (lanes
1-5). B, digitonin-permeabilized trypanosomes were
incubated with [3H]palmitoyl-CoA in the absence of
CoCl2 for 1, 5, 15, and 30 min at 37 °C (lanes
1-4). C, digitonin-permeabilized trypanosomes were
incubated with [3H]palmitoyl-CoA in the presence of 3 mM MgCl2 and 10 mM EDTA for 5 min
at 37 °C (lane 1). Subsequently, the cells were
centrifuged and resuspended in incubation buffer lacking divalent
cations and EDTA (lanes 2-5), in the presence of 3 mM MgCl2 (lanes 6-9), or in the
presence of 1 mM CoCl2 (lanes
10-13). The permeabilized cells in each condition were incubated
for 1, 5, 15, or 30 min at 37 °C. In all experiments radiolabeled
lipids were extracted and analyzed by TLC using solvent system A and
fluorography. O, origin; F, solvent front.
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Fig. 7.
Effect of DFP on lipid X formation.
Digitonin-permeabilized trypanosomes were incubated with
[3H]palmitoyl-CoA in the presence of 0, 0.1, 1, and 10 mM DFP (lanes 1-4, respectively) for 5 min at
37 °C. Radiolabeled lipids were extracted and analyzed by TLC using
solvent system A and fluorography. Lipid Y was TLC purified (lane
6) and treated with alkaline phosphatase (lane 7).
Non-radioactive lyso-phosphatidic acid standard was detected
by iodine staining and its mobility is shown.
[3H]Myristoyl-glycerol standard is shown (lane
5). Detergent present after alkaline phosphatase treatment of
lipid Y (lane 7) caused the product to migrate with an
unusual TLC mobility. O, origin; F, solvent
front.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 8.
Hypothetical model for the catabolism of
excess GPIs via lipid X. Schematic representation of lipid X
formation involving a phosphatidic acid exchange mechanism. See text
for further discussion.
It was surprising that lipid X labeled efficiently with
[3H]palmitate. Other trypanosome GPIs label specifically
with [3H]myristate, as the VSG GPI anchor contains
myristate as its sole fatty acid (39). Myristoylation occurs after GPI
biosynthesis, in a fatty acid remodeling reaction, by sequential
deacylation and reacylation (12). A second myristoylation reaction,
known as myristate exchange and for which the major substrate is VSG which already has a GPI anchor, may be a proofreading reaction to
ensure that VSG contains only myristate (23, 24). We speculate that
lipid X is palmitoylated because the use of another fatty acid allows
the myristate to be preserved for reuse. Trypanosomes cannot synthesize
myristate (35, 40, 41), and they must salvage their entire supply from
their mammalian host's bloodstream. Estimation of the concentration of
myristate in the blood indicates that there is barely enough to sustain
VSG biosynthesis when trypanosomes reach a parasitemia of over
109 cells/ml. Therefore it is essential, during GPI
metabolism, that myristate be conserved and perhaps it is even
protected from being elongated to other fatty acids (35).
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ACKNOWLEDGEMENTS |
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We thank Arthur Crossman and John Brimacombe for supplying the synthetic GlcN-PI. We also thank Angela Mehlert for assistance with the electrospray mass spectrometry studies and Igor Almeida, Lucia Güther, Terry Smith, and Karl Werbovetz for helpful discussions.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust and National Institutes of Health Grant AI 21334.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Wellcome Trust Prize Traveling Research Fellow and is currently a
Beit Memorial Fellow. To whom correspondence should be addressed. Tel.:
01382-344216; Fax: 01382-345764; E-mail:
kgmilne{at}bad.dundee.ac.uk.
The abbreviations used are:
GPI, glycosylphosphatidylinositol; PI, phosphatidylinositol; PI-PLC, PI-specific phospholipase C; GPI-PLD, GPI-specific phospholipase D; VSG, variant surface glycoprotein; GlcNAc, N-acetylglucosamine; TLC, thin layer chromatography; DFP, diisopropyl fluorophosphate; JBAM, jack bean -mannosidase; HONO, nitrous acid deamination; HPLC, high performance liquid chromatography.
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REFERENCES |
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