Glycosylphosphatidylinositol (GPI) membrane anchors are found on many eukaryotic cell-surface glycoproteins and their structures, functions, and biosynthesis have been extensively reviewed (Englund, 1993; McConville and Ferguson, 1993; Stevens, 1995; Medof, 1996; Kinoshita, 1997). The minimum GPI-anchor structure is NH2CH2CH2-HPO4-6Man[alpha]1-2Man[alpha]1-6Man[alpha]1-4GlcN[alpha]1-6myo-inositol-1-HPO4-lipid (EtNP-Man3GlcN-PI), where the lipid may be diacylglycerol, alkylacyl-glycerol or ceramide (McConville and Ferguson, 1993).
Proteins destined to be GPI anchored are attached to the amino group of the terminal ethanolamine phosphate (EtNP) residue by a transamidation reaction whereby a hydrophobic COOH-terminal peptide is exchanged for a preassembled GPI precursor in the endoplasmic reticulum (Udenfriend and Kokudula, 1995). The minimum GPI-anchor structure referred to above is highly conserved, but it may be modified with additional ethanolamine phosphate groups and with carbohydrate side-chains in a species- and tissue-specific manner (McConville and Ferguson, 1993).
The biosynthesis of GPI anchors involves the addition of GlcNAc to phosphatidylinositol (PI), to give GlcNAc-PI, which is de-N-acetylated to form GlcN-PI (Doering et al., 1989; Hirose et al., 1991; Stevens, 1993). De-N-acetylation is a prerequisite for the mannosylation of GlcN-PI to form later GPI intermediates (Smith et al., 1996; Sharma et al., 1997) and is possibly regulated in mammalian cells by GTP (Stevens, 1993). Rat GlcNAc-PI de-N-acetylase has been cloned recently, by complementation of PIG-L mutant CHO cells, and yeast and human homologs have been identified (Nakamura et al., 1997).
The GlcNAc-PI de-N-acetylase of Trypanosoma brucei has been partially purified and characterized (Milne et al., 1994) using a series of GlcNAc-PI analogues (Cottaz et al., 1993, 1995). That study concluded that (1) the fatty acid esters assist in the presentation of the substrate to the enzyme but are not essential for substrate recognition, (2) a phosphodiester linkage between the myo-inositol and glycerol residues is essential for substrate recognition, (3) product inhibition by GlcN-PI and acetate is negligible, and (4) d-myo-inositol can be replaced by l-myo-inositol without loss of substrate recognition. A more recent study using intact cell-free systems examined the substrate specificities of T.brucei and human (HeLa cell) GlcNAc-PI de-N-acetylases with respect to the size of the N-acyl (R) group that can be cleaved from a series of GlcNR-PI substrates and the ability of these enzymes to de-N-acetylate mannosylated GlcNAc-PI intermediates (Sharma et al., 1997). It was concluded that (1) the trypanosomal and human enzymes are active on GlcNR-PI substrates where R is acetyl or propionyl but much less active on substrates where R is butyryl, isobutyryl, pentanoyl, or hexanoyl and (2) mannosylated GlcNAc-PI structures are not substrates for the trypanosomal or human de-N-acetylases, demonstrating that de-N-acetylation must precede mannosylation in GPI biosynthesis.
We now compare the activities of the trypanosomal and human (HeLa cell) de-N-acetylases in intact cell-free systems using novel substrate analogues containing l-myo-inositol and 2-O-alkylated d-myo-inositol and demonstrate that the parasite and human enzymes show significant differences.
The structures of the synthetic substrates and substrate analogues (Cottaz et al., 1993, 1995; Crossman et al., 1997) used in this study are shown in Figure
Figure 1. Structures of the GlcNAc-PI substrate and substrate analogues. Trypanosome and HeLa membranes release [14C]acetate from synthetic GlcN[14C]Ac-PI
The time-dependent release of radiolabel from GlcN[14C]Ac-PI catalyzed by trypanosome and HeLa membranes is shown in Figure
Figure 2. Release of [14C]acetate from N-[14C]acetylated substrates by trypanosome and HeLa cell membranes. Trypanosome (A) andHeLa cell (B) membranes were incubated with GlcN[14C]Ac-PI (squares), GlcN[14C]Ac-P(l)I (diamonds), GlcN[14C]Ac-(2-O-Me)PI (solid circles), and GlcN[14C]Ac-(2-O-Oct)PI (triangles) for various times, and the released [14C]acetate was measured. The means of triplicate determinations (±SEM) are shown; where no error bars are shown, errors were too small to display graphically. As a negative control, boiled trypanosome or HeLa membranes were used with GlcN[14C]Ac-PI (crosses). The trypanosome de-N-acetylase will act on GlcNAc-PI containing l-myo-inositol
In order to assess the stereospecificity of the de-N-acetylasewith respect to the myo-inositol residue, a GlcN[14C]Ac-PI analogue containing l-myo-inositol instead of d-myo-inositol, GlcN[14C]Ac-P(l)I, was synthesized and carefully adjusted to the same specific activity as GlcN[14C]Ac-PI. The d- and l-myo-inositol-containing substrates were incubated with the trypanosome cell-free system and the release of [14C]acetate was measured (Figure The trypanosome GPI mannosyltransferases will act on l-myo-inositol-containing GPI intermediates
Another assay for de-N-acetylation was used to assess the substrate stereospecificity of the de-N-acetylase. In this assay, exogenous substrates or substrate analogues were added to the cell-free system in the presence of GDP-[3H]Man. Since de-N-acetylation is a strict prerequisite for mannosylation (Smith et al., 1996; Sharma et al., 1997), the formation of [3H]manno-sylated GPI intermediates may be used as an indirect measure of de-N-acetylation. With only GDP-[3H]Man present (Figure
Figure 3. Trypanosome, but not HeLa cell, membranes can de-N-acetylate and [3H]mannosylate GlcNAc-P(l)I. (A) Trypanosome membranes were labeled with GDP-[3H]Man in the presence of NEM alone (lane 1) and together with GlcN-PI (lane 2), GlcNAc-PI (lane 3), GlcN-P(l)I (lane 4) and GlcNAc-P(l)I (lane 5). The extracted glycolipids were analyzed by HPTLC and fluorography. The bands marked DPM and A[prime] are Dol-P-Man and the GPI intermediate glycolipid A[prime], respectively. (B) Glycolipids labeled in the presence of GlcNAc-P(l)I were treated with (+) and without (-) PI-PLC. The products that partitioned into butan-1-ol were analyzed by HPTLC and fluorography. (C) HeLa cell membranes were labeled with GDP-[3H]Man alone (lane 1) and together with GlcNAc-PI (lane 2), GlcN-P(l)I (lane 3) and GlcNAc-P(l)I (lane 4). The extracted glycolipids were analyzed by HPTLC and fluorography. The band marked DPM is Dol-P-Man and those marked H2 and H5 are Man1GlcN-(acyl)PI and EtN-P-Man1GlcN-(acyl) PI, respectively. The labeled GPI intermediates based on endogenous GlcN-(acyl)PI acceptors are indicated by endo, and those based on the exogenous synthetic GlcN-PI acceptor are indicated by exog.B
Previous studies have demonstrated that GlcN-P(l)I is not a substrate for the trypanosome GPI mannosyltransferase enzymes (Smith et al., 1996). However, the foregoing data demonstrate that trypanosome GPI mannosyltransferase enzymes can mannosylate a substrate containing an l-myo-inositol residue if it is presented via the de-N-acetylase enzyme. These observations strengthen the suggestion that substrate channeling occurs between GlcNAc-PI de-N-acetylase and Dol-P-Man:GlcN-PI [alpha]1-4 mannosyltransferase in trypanosomes (Smith et al., 1996).
A
B
HeLa cell de-N-acetylase will not act on GlcNAc-PI containing l-myo-inositol
In contrast to the results with the trypanosome membranes, HeLa cell membranes did not release [14C]acetic acid from GlcN[14C]Ac-P(l)I (Figure
Substrate analogues reveal differences between the trypanosome and HeLa de-N-acetylase enzymes
A previous study (Smith et al., 1997) showed that the trypanosome cell-free system is capable of de-N-acetylating GlcNAc-(2-O-Me)PI, as judged by the de-N-acetylation/[3H]mannosylation assay. However, we were unable to assess the de-N-acetylation of this compound in the HeLa cell-free system because GlcN-(2-O-Me)PI is not a substrate for the HeLa cell GPI [alpha]-mannosyltransferases. In order to address this problem, we prepared GlcN[14C]Ac-(2-O-Me)PI and a novel substrate analogue, GlcN[14C]Ac-(2-O-Oct)PI, and measured the release therefrom of [14C]acetate catalyzed by the trypanosome and HeLa cell-free systems. Both compounds are substrates for the trypanosome de-N-acetylase, exhibiting turnover rates of about 60% of that measured with GlcN[14C]Ac-PI (Figure
De-N-acetylation of GlcNAc-(2-O-Oct)PI by the trypanosome cell-free system was also evident from the de-N-acetylation/[3H]mannosylation assay (Figure
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Figure 4. Trypanosome, but not HeLa cell, membranes can de-N-acetylate and [3H]mannosylate GlcNAc-(2-O-Oct)PI. (A) Trypanosome membranes were labeled with GDP-[3H]Man in the presence of NEM alone (lane 1) and together with GlcNAc-PI (lane 2) and GlcNAc-(2-O-Oct)PI (lane 3). The extracted glycolipids were analyzed by HPTLC and fluorography. The bands marked DPM, M1-3, aM3, and A[prime] are Dol-P-Man, Man1-3GlcN-PI, Man3GlcN-(acyl)PI, and EtN-P-Man3GlcN-PI, respectively. The bands marked M1-3(Oct) are Man1-3GlcN(2-O-Oct)PI. (B) HeLa cell membranes were labeled with GDP-[3H]Man alone (lane 1) and together with GlcNAc-PI (lane 2), GlcN-(2-O-Oct)PI (lane 3), and GlcNAc-(2-O-Oct)PI (lane 4). The extracted glycolipids were analyzed by HPTLC and fluorography. The arrow indicates the expected position of EtN-P-Man1GlcN-(octyl)PI. The band marked DPM is Dol-P-Man and those marked H2 and H5 are Man1GlcN-(acyl)PI and EtN-P-Man1GlcN-(acyl)PI, respectively. Labeled GPI intermediates based on endogenous GlcN-(acyl)PI acceptors are indicated by endo and those based on the exogenous synthetic GlcN-PI acceptor are indicated by exog.
Unlike GlcNAc-PI (Figure
Conformational aspects
The reduced rates of turnover of GlcNAc-(2-O-Me)PI, compared with GlcNAc-PI, by the trypanosomal and human enzymes might be due to subtle changes that occur in PI conformation when the hydrogen bond between the hydroxyl group attached to C2 of the d-myo-inositol residue and the pro-R oxygen of the phosphodiester group is disrupted by methylation, as described recently for (2-O-methyl)PI (Zhou et al., 1997). So too the inability of the human enzyme to cleave GlcNAc-(2-O-Oct)PI might be due to conformational or steric effects.
In an attempt to understand better the stereospecificity ofthe trypanosomal and human de-N-acetylases, ROESY spectraof synthetic d-GlcNAc[alpha]1-6d-myo-inositol-1-PO4H-glycerol (GlcNAc-d-Ins-P-gro) and d-GlcNAc[alpha]1-6l-myo-inositol-1-PO4H-glycerol (GlcNAc-l-Ins-P-gro) were obtained (data not shown). Both spectra showed strong NOEs between GlcNAc H1 and myo-inositol H6 and weaker NOEs between GlcNAc H1 and myo-inositol H5. In agreement with these through-space connectivities, simulated annealing and molecular dynamics simulations suggested that GlcNAc-d-Ins-P-gro adopts a low-energy conformation in which the N-acetamido group and the phosphoglycerol moiety are cis with respect to the glycosidic bond (Figure
Figure 5. Predicted conformations of GlcNAc-d-Ins-P-gro and GlcNAc-l-Ins-P-gro. (A) The lowest energy conformer of GlcNAc-d-Ins-P-gro resulting from simulated annealing. (B) Thelowest energy conformer of GlcNAc-l-Ins-P-gro resulting from simulated annealing. (C) A representative model of the alternative conformer of GlcNAc-l-Ins-P-gro from the same simulated annealing protocol used to produce (B). For clarity, only one C atom of the glycerol moiety is shown in each case. Marked on each are the distances (in Ångstroms) between the nitrogen atom (N) of the GlcNAc residue and the phosphorus atom (P) of the phosphodiester group and between N and the oxygen atom (O) of the 4-OH of the GlcNAc residue as well as the size of the angle P-N-O.
Figure 6. Evolution of the glycosidic angles during 1 ns of unrestrained molecular dynamics. Plots of the behavior of the glycosidic dihedral angles [phis] and [psi] during the last 1 ns of a 1.02 ns unrestrained molecular dynamics simulation. From left to right, [phis] versus [psi], [phis] versus time and [psi] versus time for GlcNAc-d-Ins-P-gro (A)and GlcNAc-l-Ins-P-gro (B).
Conclusions
We have already shown that trypanosomal and human (HeLa cell) GlcNAc-PI de-N-acetylases have similar specificities with respect to the size of the N-acyl group (R) that can be removed from a series of GlcNR-PI substrates. In this paper we have examined the abilities of the human and parasite enzymes to act on substrates with O-alkyl substituents attached to position 2 of the d-myo-inositol residue and their stereospecificities towards the myo-inositol residue. The results show that the human enzyme is more fastidious than the parasite enzyme on both counts. Thus, whereas the trypanosomal enzyme turned over all three of the substrate analogues investigated (i.e., GlcNAc-(2-O-Me)PI, GlcNAc-(2-O-Oct)PI and GlcNAc-P(l)I), albeit at lower rates than for GlcNAc-PI, the human enzyme acted only on GlcNAc-(2-O-Me)PI at a detectable rate. These differences in the substrate specificities of the trypanosomal and human GlcNAc-PI de-N-acetylases suggest that it might be possible to design or discover parasite-specific de-N-acetylase inhibitors of therapeutic potential.
Materials
GDP-[2-3H]Man (15.0 Ci/mmol), [3H]acetate (135.4 mCi/mM) and En3Hance were purchased from Dupont-NEN. [14C]Acetic anhydride (112 mCi/mmol) was purchased from Amersham, IST C8 Isolute reverse-phase cartridges from Crawford Scientific, Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) from Oxford GlycoSystems and jack bean [alpha]-mannosidase (JBAM) from Boehringer. Human serum was used as a source of GPI-specific phospholipase D (GPI-PLD). All the other reagents were purchased from Sigma.
Preparation of trypanosomes and the trypanosome cell-free system
Bloodstream forms of Trypanosoma brucei (variant MITat1.4) were isolated from infected rats and mice. Trypanosome membranes (trypanosome cell-free system) were prepared as described previously by Masterson et al. (1989), except that the cells were not preincubated with tunicamycin prior to lysis.
Preparation of HeLa cells and the HeLa cell-free system
HeLa cells were grown at 37°C in Dulbecco's high-glucose modified minimal essential medium supplemented with 10% fetal calf serum, 1% nonessential amino acids, 1 mM glutamine, penicillin, and streptomycin in a 5% CO2 atmosphere. The HeLa cell-free system was prepared as described previously (Sharma et al., 1997).
Synthetic substrates and substrate analogues
d-GlcN[alpha]1-6d-myo-inositol-1-HPO4-(sn-1,2-dipalmitoylglycerol) (GlcN-PI) and d-GlcN[alpha]1-6l-myo-inositol-1-HPO4 (sn-1,2-dipalmitoylglycerol) (GlcN-P(l)I) were prepared as described previously (Cottaz et al., 1993) as were d-GlcN[alpha]1-6(2-O-methyl-d-myo-inositol)-1-HPO4-(sn-1,2-dipalmitoylglycerol)(GlcN-(2-O-Me)PI) (Crossman et al., 1997), d-GlcN[alpha]1-6(2-O-octyl-d-myo-inositol-1-HPO4)-(sn-1,2-dipalmitoylglycerol) (GlcN-(2-O-Oct)PI) (Crossman et al., 1997), d-GlcN[alpha]1-6d-myo-inositol-1-HPO4-(sn)-3-glycerol (GlcN-d-Ins-P-gro) (Cottaz et al., 1995), and d-GlcN[alpha]1-6l-myo-inositol-1-HPO4-(sn)-3-glycerol)(GlcN-l-Ins-P-gro) (Cottaz et al., 1995). Nonradioactive N-acetylations were performed at 0°C in 100 µl of saturated NaHCO3 by the addition of three aliquots (2.5 µl) of acetic anhydride at 10 min intervals. The reaction mixture was allowed to warm to room temperature and the N-acetylated glycolipids were then isolated and desalted by extraction into butan-1-ol.
The purity of the synthetic compounds (both before and after N-acetylation) and the concentrations of stock solutions were assessed by negative-ion electrospray mass spectrometry and gas chromatography-mass spectrometry, as described previously (Sharma et al., 1997).
N-[14C]Acetylation and purification of substrates and substrate analogues
Synthetic GlcN-PI substrates and substrate analogues were N-acetylated with [14C]acetic anhydride as described for GlcN-PI: 1 ml of GlcN-PI (0.2 mg/ml in tetrahydrofuran/methanol (1:1, v/v)) was mixed with 50 µl of pyridine and 4.5 µmol of [14C]acetic anhydride (added as a 5% w/v solution in dry toluene). After 20 min at room temperature, 10 µl of acetic anhydride was added to the mixture which was left for a further 1 h to ensure complete N-acetylation. The reaction mixture was adjusted to 5 ml with 5% propan-1-ol in 100 mM ammonium acetate and loaded onto a pre-equilibrated 500 mg C8 Isolute cartridge. The cartridge was then washed with ~175 ml of 5% propan-1-ol in 100 mM ammonium acetate, i.e., until the eluting [14C]-radioactivity fell to background levels. Stepwise elution with 8 ml each of 20%, 40%, 60%, and 80% propan-1-ol in 100 mM ammonium acetate was then performed. A major radiolabeled fraction eluted at 40% propan-1-ol in 100 mM ammonium acetate which, when analyzed by ES-MS, was shown to contain GlcN[14C]Ac-PI. The concentration of the purified GlcN[14C]Ac-PI was measured by determining its myo-inositol content by GC-MS (Sharma et al., 1997). The specific activity of the substrate was adjusted to 16 mCi/mmol by the addition of nonradioactive GlcNAc-PI.
Trypanosome and HeLa de-N-acetylase assay using GlcN[14C]Ac-PI substrates
Washed trypanosome (5 × 107 cell equivalents/ml) and HeLa(2 × 107 cell equivalents /ml) membranes (cell-free systems) were added to tubes containing dried GlcN[14C]Ac-PI to give a final substrate concentration of 3.1 µM or 1.55 µM, respectively. The mixtures were then incubated at 30°C and 37°C, respectively. Triplicate (or greater) samples of 50 µl or 100 µl, respectively, were taken at each time point when the reactions were terminated by the addition of 50 µl of propan-1-ol, vortexing, and snap-freezing. The samples were then thawed and mixed with 100 µl of 1 M ammonium acetate and 800 µl of water. After centrifugation in a microfuge, the supernatant was passed through a pre-equilibrated 100 mg C8 cartridge followed by 1 ml of 5% propan-1-ol in 100 mM ammonium acetate. The entire eluate containing any released [14C]acetate was collected and counted for radioactivity.
Trypanosome and HeLa de-N-acylation/[3H]mannosylation assays
These assays, which rely on the [3H]mannosylation of GlcN-PI (and its analogues) generated by the de-N-acetylation of GlcNAc-PI (and its analogues), have been described previously (Sharma et al., 1997).
HPTLC
Samples and glycolipid standards were applied to 10 or 20 cm aluminum-backed silica gel 60 HPTLC plates (Merck) which were developed in chloroform/methanol/1 M ammonium acetate/13 M ammonium hydroxide/water (180:140:9:9:23, v/v). Radiolabeled components were detected after spraying with En3Hance by fluorography at -70°C using Kodak XAR-5 film and an intensifying screen.
PI-PLC digests
Samples (~4 nmol) of synthetic GlcN-PI and GlcN-P(l)I were dried under a stream of nitrogen and redissolved in 20 µl of digestion buffer (25 mM Tris-acetate, pH 7.4, 0.1% Na-deoxycholate) containing 0.04 units of B.thuringiensis PI-PLC. After 1.5 h at 37°C, the products were partitioned between water and butan-1-ol whereafter each phase was analyzed for myo-inositol content by GC-MS. The presence of myo-inositol in the aqueous phase indicated PI-PLC cleavage of the glycolipid substrate.
GPI headgroup analysis
Radiolabeled GPI species were purified by HPTLC and then subjected to basic hydrolysis, nitrous acid deamination, NaBH4 reduction and aq. HF dephosphorylation as described previously (Smith et al., 1997). This procedure generates radiolabeled neutral glycans terminating in 2,5-anydromannitol (AHM) which are analyzed by Bio-Gel P4 gel filtration in order to assign the number of Man residues in the GPI species (Smith et al., 1997).
NMR and molecular modeling
Samples (5 mg) of synthetic d-GlcNAc[alpha]1-6d-myo-inositol-1-PO4H-glycerol (GlcNAc-d-Ins-P-gro) and d-GlcNAc[alpha]1-6l-myo-inositol-1-PO4H-glycerol (GlcNAc-l-Ins-P-gro), prepared by N-acetylation of the corresponding amines (Cottaz et al., 1995), were dissolved in 2H2O and NMR spectra were recorded at 500.13 MHz and 303 K using a Varian Unity Plus spectrometer. Two-dimensional 1H-1H COSY experiments consisted of 512 t1 increments, each of 8 transients, and 1024 complex points in t2. Homonuclear 1H ROESY experiments were carried out according to Homans and Forster (1992) with 512 t1 increments, each of 8 transients, and 1024 complex points in t2 and a 350 ms spin-lock period. Spectra were analyzed with Varian VNMR software and were zero-filled in each dimension. COSY and ROESY spectra were apodized using sine-bell and cosine-bell functions, respectively, in each dimension prior to Fourier transformation. Cross-peak volumes were integrated to provide ROE intensities.
Molecular modeling was carried out on Silicon Graphics computers. Structures were constructed and analyzed with the molecular visualization package Insight II as well as with the molecular modeling package DISCOVER (Molecular Simulations, San Diego, CA). Simulations were performed in vacuo with a dielectric constant of 80.0 using the AMBER force field (Weiner et al., 1984) with the carbohydrate parameters described by Homans (1990) and with all of the torsional terms corresponding to the exo-anomeric effect set to zero (Rutherford et al., 1993). Restrained simulated annealing and unrestrained molecular dynamics simulations were carried out as described previously (Rutherford et al., 1995). Fifty annealed structures were generated, and the lowest-energy structure was used in a 1020 pS molecular dynamics simulation at 300 K. The glycosidic dihedral angles [phis] and [psi] in Figure
This work was supported by a programme grant from the Wellcome Trust. D.K.S. thanks the MRC for a Ph.D. studentship.
AHM, 2,5-anydromannitol; GPI, glycosylphosphatidylinositol; GPI-PLD, GPI-specific phospholipase D; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; GlcNAc-PI, N-acetylglucosaminyl-phosphatidylinositol.
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