A Novel Glycosylphosphatidylinositol in African Trypanosomes
A POSSIBLE CATABOLIC INTERMEDIATE*

Kenneth G. MilneDagger , Michael A. J. Ferguson, and Paul T. Englund§

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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).

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 -70 °C.

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 -20 °C and dried into tubes as required.

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 alpha -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.

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-alpha -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).

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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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).


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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.

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.


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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.

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).


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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.

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 - 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-GlcNalpha 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).

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).


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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.

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. 


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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

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.


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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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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 alpha -mannosidase; HONO, nitrous acid deamination; HPLC, high performance liquid chromatography.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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