Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis

Deepak K. Sharma4, Terry K. Smith, Charles T. Weller2, Arthur Crossman1, John S. Brimacombe1 and Michael A.J. Ferguson3

Division of Molecular Parasitology & Biological Chemistry, Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland, 1Department of Chemistry, University of Dundee, Dundee DD1 4HN, Scotland and 2School of Biomedical Sciences, University of St. Andrews, St. Andrews KY16 9ST, Scotland

Received on August 6, 1998; revised on September 9, 1998; accepted on September 14, 1998

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d-myo-inositol or on the GlcNAc-PI diastereoisomer containing l-myo-inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d-myo-inositol component was also seen for the trypanosomal GPI [alpha]-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAc[alpha]1-6d-myo-inositol-1-HPO4-(sn)-3-glycerol and its diastereoisomer d-GlcNAc[alpha]1-6l-myo-inositol-1-HPO4-(sn)-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the [alpha]-mannosyltransferases in this conformation.

Key words: GlcNAc-PI/GPI/biosynthesis/mannosylation/de-N-acetylase

Introduction

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.

Results and discussion

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 1.


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 2. In both cases, the released label was analyzed by strong anion exchange HPLC and was shown to co-elute with a standard of [3H]acetic acid (data not shown), confirming that the release was due to a de-N-acetylase activity rather than to exoglycosidase or phospholipase activity. The release of [14C]acetate from synthetic GlcN[14C]Ac-PI represents a new and convenient assay for GlcNAc-PI de-N-acetylase activity.

   A
   B

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 2A). It is clear that, under these conditions, GlcN[14C]Ac-P(l)I is turned over at approximately one fifth of the rate of GlcN[14C]Ac-PI. This result is qualitatively similar to, but quantitatively different from, that previously reported (Milne et al., 1994) which showed that the l-myo-inositol-containing analogue was turned over at approximately the same rate as GlcN[14C]Ac-PI. However, in that study a detergent-solubilized, partially purified (and much less active) enzyme preparation was used rather than the washed membranes.

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 3A, lane 1) only dolichol-phosphate-mannose (Dol-P-Man) and a small amount of endogenous GPI intermediates are radiolabeled. As previously reported (Smith et al., 1996; Sharma et al., 1997), the addition of exogenous GlcN-PI and GlcNAc-PI stimulated the formation of [3H]mannosylated GPI intermediates (Figure 3A, lanes 2 and 3) whereas the addition of GlcN-P(l)I did not (Figure 3A, lane 4). However, the addition of GlcNAc-P(l)I did stimulate the formation of [3H]mannosylated glycolipids(Figure 3A, lane 5). Analysis of the mannosylated species showed them to be sensitive to nitrous acid deamination (data not shown), indicating that they were de-N-acetylated GPI intermediates. However, unlike most of the mannosylated GPI intermediates produced in the presence of GlcNAc-PI, those produced in the presence of GlcNAc-P(l)I were PI-PLC resistant (Figure 3B, lanes 1 and 2). The stereospecificity of B.thuringiensis PI-PLC was established following digestion of synthetic GlcN-PI and GlcN-P(l)I with the enzyme, partitioning the products between water and butan-1-ol and analyzing each of the phases for myo-inositol content by GC-MS (data not shown). Since these data showed that B.thuringiensis PI-PLC is specific for GlcN-PI containing d-myo-inositol, it follows that the PI-PLC-resistant, [3H]mannosylated GPI intermediates produced from GlcNAc-P(l)I must contain l-myo-inositol.

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).

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 2B). In the indirect de-N-acetylation/[3H]mannosylation assay, an endogenous H5 species (EtNP-Man1GlcN-(acyl)PI) was labeled in the presence of GDP-[3H]Man (Figure 3C, lane 1) and, as previously described (Sharma et al., 1997; Smith et al., 1997), the addition of exogenous GlcN-PI or GlcNAc-PI resulted in the labeling of exogenous H2 and H5 bands (Figure 3C, lane 2). However, GlcN-P(l)I did not stimulate the labeling of exogenous H2- and H5-like glycolipids (Figure 3C, lane 3), suggesting that the HeLa cell-free system cannot acylate the inositol ring of GlcN-P(l)I and/or subsequently mannosylate the GlcN residue. Therefore, the indirect de-N-acetylation/[3H]mannosylation assay cannot be used to assess the de-N-acetylation of GlcNAc-P(l)I in this case.

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 2A). Whereas GlcN[14C]Ac-(2-O-Me)PI was also de-N-acetylated by the HeLa cell free system, though with lower efficiency, no turnover of GlcN[14C]Ac-(2-O-Oct)PI was detected (Figure 2B).

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 4A), which showed that the addition of GlcNAc-(2-O-Oct)PI stimulated the labeling of one major component (Figure 4A, lane 3). This glycolipid, which has an Rf similar to that of Man1GlcN-PI, was shown to be PI-PLC-resistant, nitrous acid-sensitive and jack bean [alpha]-mannosidase-sensitive, and to produce the headgroup derivative Man2AHM (data not shown). Taken together, these data suggest Man2GlcN-(2-O-Oct)PI as the most likely structure for this glycolipid. However, whereas GlcNAc-(2-O-Me)PI is manno-sylated to a similar extent to GlcNAc-PI (Smith et al., 1997), GlcNAc-(2-O-Oct)PI appears to be a relatively poor substrate for the GPI pathway [alpha]-mannosyltransferases (Figure 4A, compare lanes 2 and 3), despite the fact that both substrate analogues appear to be de-N-acetylated at similar rates (Figure 2A). Furthermore, the GlcNAc-(2-O-Oct)PI that is processed appears to accumulate as Man2GlcN-(2-O-Oct)PI, suggesting that Man2GlcN-(2-O-Oct)PI is a poor substrate for the third [alpha]-manno-syltransferase.

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 4B, lane 2), GlcNAc-(2-Oct)PI was unable to stimulate the labeling of GPI intermediates in the HeLa cell de-N-acetylation/[3H]mannosylation assay (Figure 4B, lane 4). Since GlcN-(2-O-Oct)PI was also unable to generate [3H]mannosylated products, suggesting that the C8 alkyl chain on the 2-position of the d-myo-inositol residue cannot substitute for the C16 acyl chain present on the natural HeLa cell substrate (Sevlever et al., 1995), the indirect de-N-acetylation/[3H]mannosylation assay cannot be used to assess de-N-acetylation in this case.

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 5A), whereas these groups are trans in GlcNAc-l-Ins-P-gro (Figure 5B). However, unrestrained molecular dynamic simulations suggest that GlcNAc-l-Ins-P-gro is able to adopt a cis conformation for significant periods of time (Figure 6), thus mimicking the structure of the d-isomer with respect to the vector between the nitrogen atom (N) of GlcNAc and the phosphorus atom (P) of the phosphodiester (Figure 5C). This is significant because the phosphate group is known to be important for substrate recognition by the trypanosome de-N-acetylase (Milne et al., 1994) and, therefore, the vector between the phosphate and the GlcN nitrogen atom (the site of de-N-acetylation) is likely to be an important determinant for substrate recognition. Thus, it is conceivable that the trypanosomal de-N-acetylase reacts with GlcNAc-P(l)I only when it adopts this alternative conformation and that the substrate is trapped in this conformation as it is channeled from the de-N-acetylase to the [alpha]-mannosyltransferases. This model predicts that the orientations of the hydroxyl groups attached to positions 2-5 of the inositol ring are not critical for substrate recognition by the trypanosomal de-N-acetylase (compare the structures in Figure 5A,C), whereas the same is not true for the human enzyme. It is worth noting that both the vector between P and the oxygen atom (O) of the 4-OH of the GlcN residue (the acceptor site for the first [alpha]-mannosyltransferase of the GPI pathway) and the P-N-O angle for GlcNAc-d-Ins-P-gro (Figure 5A) are very similar to those for the alternative conformation of GlcNAc-l-Ins-P-gro (Figure 5C). This might explain why GlcN-P(l)I emerging from the de-N-acetylase in the alternative conformation can act as a mannose-acceptor.


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 and methods

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 6 refer to the formal definitions of [phis]H and [psi]H in IUPAC nomenclature, and are defined as H1-C1-O1-Cx and C1-O1-Cx-Hx, respectively.

Acknowledgments

This work was supported by a programme grant from the Wellcome Trust. D.K.S. thanks the MRC for a Ph.D. studentship.

Abbreviations

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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3To whom correspondence should be addressed
4Present address: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.


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