Eur. J. Biochem. 267, 3385±3392 (2000) q FEBS 2000

Transient N-acetylgalactosaminylation of mannosyl phosphate side chain in Paramecium primaurelia glycosylphosphatidylinositols Nahid Azzouz1, Peter Gerold1, JoÈrg Schmidt1, Yvonne Capdeville2 and Ralph T. Schwarz1 1

Med. Zentrum fuÈr Hygiene und Medizinische Mikrobiologie, Philipps-UniversitaÈt Marburg, Germany; 2Centre de GeÂneÂtique MoleÂculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France

The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side. Keywords: cell-free system; glycosylphosphatidylinositol; GPtdIns; GPtdIns biosynthesis; Paramecium primaurelia.

Eukaryotic cells use glycosylphosphatidylinositol (GPtdIns) structures as an alternative to anchor proteins in the cytoplasmic membrane [1]. Besides their role in anchoring and targetting proteins to the apical membrane, substantial biological and immunological properties have been shown to be linked to the GPtdIns structures [2±4]. GPtdIns plays a crucial role in the biology of parasites, as their GPtdIns surface antigens have to interact with the host immune system. Furthermore, in the case of parasitic protozoa it has been shown that GPtdIns types derived from the human malaria parasite Plasmodium falciparum, are involved in the pathology of severe malaria [5,6]. The structure and biosynthesis of GPtdIns-anchor precursors have been elucidated in protozoa and mammalian cells [7±10]. An evolutionarily conserved core glycan consists of a trimannosyl glucosamine structure linked to ethanolamine phosphate: ethanolamine-PO4-6Mana1±2Mana1±6Mana1±4 GlcNH2. The basic core glycan can be modified by additional sugars or molecules such as ethanolamine Correspondence to N. Azzouz, Med. Zentrum fuÈr Hygiene und Medizinische Mikrobiologie, Philipps-UniversitaÈt Marburg, Robert-Koch-Strasse 17, 35037 Marburg, Germany. Tel.: 1 49 6421 2865149, E-mail: [email protected] Abbreviations: ATPgS, adenosine 5 00 -O-(3-thiotriphosphate); Dol-P-Man, dolichol phosphate mannose; GPtdIns, glycosylphosphatidylinositol; Gu, glucose units; HPAEC, high pH anion exchange chromatography; PtdIns, phosphatidylinositol; PtdIns-PLC, PtdIns-phospholipase C; ER, endoplasmic reticulum; BiP, Trypanosoma brucei heavy chain binding protein. (Received 26 January 2000, revised 27 March 2000, accepted 6 April 2000)

phosphate, Glc, GalNAc or Man [1,11]. Concerning the topology of GPtdIns-anchor biosynthesis it has been suggested that at least the early nonmannosylated and the mannosylated GPtdIns biosynthetic intermediates are localized at the cytoplasmic face of the endoplasmic reticulum (ER) [12±14]. As preassembled GPtdIns-anchor precursors are transferred to nascent proteins in the lumen of the ER in a transamidase-like reaction [15], an obligatory translocation of one of the intermediates into the lumen of the ER membrane is required. We used P. primaurelia as model to elucidate and to understand the biosynthetic pathways leading to the formation of protozoan GPtdIns-anchors; such knowledge will contribute to the development of new drugs and anti-parasitic vaccines. We have described the biosynthetic pathway for a polar glycolipid which is destined to anchor the P. primaurelia GPtdIns-anchored proteins [7]. The core glycan of this glycolipid is modified by mannosyl phosphate. Here, we report that the biosynthesis of this modification starts with the phosphorylation of the trimannosyl-intermediate (Man3-intermediate) followed by the mannosylation reaction involving dolichol phosphate mannose (Dol-P-Man). The phosphorylation reaction was shown to be ATP-dependent as the presence of adenosine 5 00 -O-(3-thiotriphosphate) (ATPgS), a noncleavable analogue of ATP, leads to the accumulation of the Man3-intermediate indicating that the kinase involved is ATP-dependent and substrate specific. We also report the identification of a hydrophilic intermediate containing an additional N-acetylhexosamine. The latter was identified as N-acetylgalactosamine linked to mannosyl phosphate. The GalNAc moiety is removed in the course of the GPtdIns biosynthesis as this residue is not present in the penultimate

3386 N. Azzouz et al. (Eur. J. Biochem. 267)

GPtdIns precursor having only mannosyl phosphate as side chain and is also absent in the final phosphoethanolaminecontaining GPtdIns precursor. We suggest that the transient presence of a GalNAc-containing intermediate could play a role in the translocation of the GPtdIns-precursor to the luminal side of the ER. For the first time such an oligosaccharide processing, which seems to be essential for the assembly and maturation of Paramecium GPtdIns types, is reported.

M AT E R I A L S A N D M E T H O D S Materials GDP-[3,4-3H]mannose (15.1 Ci´mmol21), UDP-N-acetyl-d[6-3H]glucosamine (20 Ci´mmol21) and UDP-N-acetyl-d[6-3H]galactosamine (20 Ci´mmol21) were from DuPont±New England Nuclear. ATP and ATPgS were from Calbiochem. Proteinase K was from Boehringer Mannheim Corp. Bacillus cereus phosphatidylinositol specific phospholipase C (PtdInsPLC) was from Oxford Glycosystems (Abingdon, UK). Antibodies raised against Trypanosoma brucei heavy chain binding protein (BiP) were a gift from J. D. Bangs (University of Wisconsin, Madison, WI, USA). The Super SignalTM West Dura kit for chemiluminescent detection of Western blots was from Pierce. Membrane preparations Cells of P. primaurelia strain 156 were grown as described [7], supplemented with 0.5 mm 2-deoxy 2-fluoro glucose and 10 mg´mL21 tunicamycin. After 1 h at 37 8C, cells were hypotonically lysed using the method described by Masterson et al. [16]. Briefly, cells were resuspended in ice-cold water containing 0.1 mm Tos-Lys-CH2Cl and 1 mg´mL21 leupeptin prior to disruption with 50 strokes in a Dounce homogenizer. Biosynthesis and extraction of GPtdIns glycolipids in vitro The lysate (106 cells) was washed three times with 50 mm Na/Hepes pH 7.4, 50 mm KCl, 10 mm MgCl2, 0.1 mm TosLys-CH2Cl, 1 mg´mL21 leupeptin (buffer A). After centrifugation (Beckman J21, 10.000 r.p.m., 20 min at 4 8C), the pellet was resuspended in buffer A supplemented with 5 mm MnCl2, 0.2 mg´mL21 tunicamycin (Calbiochem), 1 mm ATP, 1 mm CoA and 2 mCi 3H-labelled nucleotide sugars or 20 000 c.p.m. TLC-purified Dol-P-[3H]Man or 10 000 c.p.m. TLC-purified [3H]Man-labelled glycolipid II. Assays were supplemented with 1 mm GDP-mannose for experiments involving UDP-N-acetyl-[3H]glucosamine, with 1 mm UDP-N-acetylglucosamine for experiments involving GDP-[3H]mannose or both in experiments involving UDP-N-acetyl-[3H]galactosamine. Extracted glycolipids were analysed by TLC. In experiments involving ATPgS, membranes were first incubated with this component for 10 min before adding the radiolabelled nucleotide sugar.

q FEBS 2000

Generation and analysis of the core-glycans Neutral core glycans were generated from TLC-purified glycolipids by dephosphorylation, deamination and reduction as described [17]. Glycolipids were dephosphorylated by treatment with ice-cold 48% aqueous HF for 60 h at 0 8C. After neutralization with saturated LiOH, the mixture was desalted, deaminated by HNO2 treatment for 4 h, and reduced by sodium borohydride for 5 h. The resulting material was desalted on AG3WX4 (OH2) and AG50WX12 (H1) tandem ion-exchange columns and filtered through a 0.2-mm filter. Neutral glycans were analysed by high pH anion exchange chromatography (HPAEC) on a Dionex Basic Chromatography System (Dionex Corp.). The analysis was accomplished using a gradient elution program as described [17]. Monosaccharides were obtained by acid hydrolysis (4 m HCl for 4 h at 100 8C) and analysed by HPAEC, using isocratic conditions: 15 mm NaOH. Radiolabelled glycans were detected by scintillation. Elution positions of the coinjected individual glucose oligomers and monosaccharide standards were detected using a pulsed amperometric detector. Analysis of HNO2 fragments Glycolipids were deaminated by nitrous acid (HNO2) treatment, to generate hydrophilic carbohydrate fragments [18]. For HPAEC analysis the deamination buffer contained n-octylglycoside (0.5%) as a detergent instead of SDS. The generated hydrophilic fragments were analysed by Bio-Gel P4 sizeexclusion columns (1 cm  130 cm, , 400 mesh), eluted with 0.2 m ammonium acetate containing 0.02% sodium azide. Fractions (800 mL) were collected at a rate of one fraction per 24 min. Glucose oligomers, derived from partially hydrolysed dextran [19], were included as internal standards and detected by staining aliquots (25 mL) with 2 mg´mL21 orcinol in concentrated sulphuric acid. HNO2 fragments were also analysed by HPAEC using the following program: 100 mm NaOH (buffer B) for 0.6 min, an increase in buffer C (100 mm NaOH; 0.25 m sodium acetate) from 0 to 30%, and then to 50% buffer C in 15 min, and to 100% buffer C in 5 min for 15 min at a flow rate of 1 mL´min21; fractions were then collected at 0.4 min. Incorporation of Dol-P-Man and glycolipid II into liposomes Egg lecithin (8 mg) in chloroform/methanol (2 : 1, v/v) was dried to a thin film under a stream of argon. The film was redissolved in 2 mL 10 mm Tris/HCl, 100 mm NaCl pH 7.4 containing 1% Triton X-100 (buffer D). TLC-purified glycolipids or TLC-purified Dol-P-[3H]Man which had been solubilized in buffer D were added to the dispersed lipid suspension. SM-2 BioBeads (0.6 mg), pretreated as described by Holloway [20], were added and the mixture stirred for 48 h at 4 8C. SM-2 Bio-Beads were removed by filtration of the solution through glass wool.

TLC

Enzyme digestions and chemical treatments

The recovered glycolipids were dried in a Speed-Vac (Savant Inc., Holbrook, NY, USA) and partitioned between water and water-saturated n-butanol. Subsequently they were analysed on silica 60 plates (Merck) using chloroform/ methanol/0.25% KCl (10 : 10 : 3, v/v/v) as solvent. After chromatography, the plates were dried and scanned for radioactivity with a Berthold LB 2842 automatic scanner.

The water soluble fragments obtained after HNO2 deamination of glycolipid IV were subjected to calf intestine alkaline phosphatase (Boehringer-Manmheim) treatment in 25 mm Tris/HCl pH 8.9 containing 10 mm MgCl2, 0.1 mm ZnCl and 10 U enzyme. For PtdIns-PLC treatment, membranes were treated with 5 U´mL21 PtdIns-PLC for 60 min at 37 8C. Mild acid treatment was performed with 0.02 m HCl for 5 min at

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P. primaurelia GPtdIns biosynthesis (Eur. J. Biochem. 267) 3387

100 8C. Integrity of ER vesicles was determined by protease protection experiments using BiP, an ER luminal protein. Protease K treatment was undertaken both with (0.5%) Triton X-100 and without, according to Vidugiriene and Menon [13]. Proteins were analysed by 10% SDS/PAGE, transferred to nitrocellulose and probed with specific monoclonal antibodies, which had been raised against T. brucei BiP and chemiluminescent peroxidase substrate supplied with the Super SignalTM West Dura kit. The blots were exposed to X-ray film.

The poor labelling with Dol-P-[3H]Man could be attributed to the presence of detergent (Triton X-100) [27]. To overcome this problem and to maximize the labelling of GPtdIns, Dol-P[3H]Man solubilized in Triton X-100 was mixed with phospholipids and the detergent was removed as described, leading to the formation of vesicles containing Dol-P-[3H]Man. Vesicles were first fused with membranes for 10 min at 37 8C. Glycolipids were extracted in one step with C/M/W (10 : 10 : 3, v/v/v) after 60 min incubation and analysed by TLC. Although the labelling of the GPtdIns molecules was lower (Fig. 1C), the TLC profile is different from that obtained following direct labelling with Dol-P-[3H]Man solubilized in detergent, as we were now able to label the polar glycolipid II.

R E S U LT S GPtdIns glycolipids synthesized in vitro by P. primaurelia We have shown previously that the core glycans of the polar GPtdIns glycolipids of P. primaurelia are modified by mannosyl phosphate, which is linked to the mannose adjacent to the nonacetylated glucosamine molecule [7]. Our data suggest that the mannosyl phosphate modification is added to the trimannosyl core glycan before the transfer of the GPtdIns precursor onto the polypeptide chain. Therefore we investigated the biosynthesis of the mannosyl phosphate modification. The known donors for mannosylation reactions are Dol-P-Man and/or GDP-Man. The biosynthesis of the mannosyl phosphate side chain in P. primaurelia may have a direct implication for the membrane topology of the assembly process during GPtdIns biosynthesis. In contrast with Dol-P-Man which is available on both sides of the ER, GDP-Man is available only on the cytoplasmic side [21±23]. To discriminate between GDP-Man and Dol-P-Man, GPtdIns glycolipids were labelled in vitro using P. primaurelia membranes with GDP-[3H]Man or TLC-purified Dol-P-[3H]Man. Membranes were prepared from cells preincubated with tunicamycin and 2-d-fluoroglucose, to inhibit the N-glycosylation pathway and to block the endogenous formation of Dol-P-Man and dolichol phosphate glucose, respectively [24]. This leads to better incorporation of [3H]Man into GPtdIns molecules. The labelling was stopped by adding C/M (1 : 1, v/v) to the incubation mixture to achieve a final concentration of C/M/W (10 : 10 : 3, v/v/v). TLC analysis, using silica 60 plates, showed a spectrum of glycolipids (I to VII) and Dol-P-Man (Fig. 1A) labelled with GDP-[3 H]Man. Glycolipids I to VII were also labelled with UDP-N-acetyl-[3H]glucosamine (data not shown). All glycolipids have been structurally identified as glycosylinositol phosphoceramides [7]. Dol-P-Man was identified by its chromatographic mobility, its sensitivity to mild acid treatment as well as by the inhibition of its formation using amphomycin (data not shown). Dol-P-Man is the donor of mannnose residues TLC-purified Dol-P-[3H]Man was incubated with membranes in the presence of amphomycin known to block the synthesis of Dol-P-Man from GDP-Man [25]. In contrast with labelling with GDP-[3H]Man, the efficiency of labelling using Dol-P[3H]Man was poor (Fig. 1B). By comparison with the chromatographic mobility of glycolipids in standard incubation with GDP-[3 H]Man (Fig. 1A), glycolipid IV is the major labelled GPtdIns intermediate. However, the early GPtdIns intermediates (glycolipids V±VII) could be detected following TLC chromatography but not the polar glycolipids (I±III). In T. brucei, Dol-P-Man is the donor of the trimannosyl core glycan of GPtdIns molecules [26]. As early P. primaurlia GPtdIns (Man1 to Man3-intermediates) glycolipids are labelled in the presence of Dol-P-[3H]Man we suggest that Dol-P-Man is also the donor of these mannose residues.

Fig. 1. TLC analysis of the P. primaurelia GPtdIns glycolipids. Glycolipids were labelled in vitro using P. primaurelia membranes with GDP-[3H]Man (A), Dol-P-[3H]Man solubilized in Triton X-100 (B) or DolP-[3H]Man-containing vesicles (C). The labelled glycolipids were extracted with C/M/W (10 : 10 : 3, v/v/v). The C/M/W extracted glycolipids were dried and partitioned between water and water-saturated n-butanol. Glycolipids recovered in the butanol phase were analysed on silica TLC plates using solvent system C/M/0.25% KCl (10 : 10 : 3, by volume). They were then scanned for radioactivity using a TLC scanner (Berthold LB 2842). O, Origin; F, front. The structure of the previously characterized glycolipids is indicated in (A) E, Ethanolamine phosphate; M, mannose; P; phosphate; GIPC; glucosamine inositol-phosphoceramide.

3388 N. Azzouz et al. (Eur. J. Biochem. 267)

q FEBS 2000

from glycolipid IV eluted later than the corresponding fragment generated from glycolipid III indicating a difference in their charges. One explanation could be that glycolipid IV is the phophorylated Man3-intermediate. Therefore we subjected the HNO2 fragment generated from glycolipid IV to alkaline

Fig. 2. Bio-Gel P4 and Dionex-HPAEC analysis of the hydrophilic core glycans generated from glycolipids II, III and IV. TLC-purified [3H]Manlabelled glycolipids were deaminated with nitrous acid. The hydrophilic moieties generated were sized on a Bio-Gel P4 column (A±C). The elution positions of the coinjected glucose oligomer standards are indicated at the top of (A). (D±E) TLC-purified [3H]Man-labelled glycolipids II and IV were dephosphorylated, deaminated and reduced. The resulting neutral glycans were analysed by Dionex-HPAEC. The elution position of the coinjected glucose oligomer standards and Man3-AHM standard generated from glycolipid III are indicated at the top of (D). IP, injection peak.

Characterization of glycolipids II and IV core glycans We generated the hydrophilic moieties of glycolipids II and IV by deamination (HNO2) and the neutral glycans obtained by dephosphorylation, deamination and reduction. The analysis by Bio-Gel P4 size exclusion chromatography showed that the water-soluble HNO2 fragments generated from glycolipids II, III and IV eluted at 12, 8.5 and 8 glucose units (Gu), respectively (Fig. 2A±C). The data suggest that the fragment generated from glycolipid II is larger or contains an additional charge compared with the structure of a corresponding fragment generated from glycolipid III, which has the following structure: Man-Man-(P-Man)Man-anhydromannose [7]. Dionex-HPAEC analysis of the neutral glycans generated from glycolipids II and IV showed that fragments from both GPtdIns types coeluted with the Man3-AHM fragment generated from glycolipid III suggesting an identical core glycan (Fig. 2D,E). Glycolipid IV is the phosphorylated Man3-intermediate Because the difference in the elution position on the P4 column between the HNO2 fragments generated from glycolipids III and IV is minimal, we compared their elution positions on Dionex-HPAEC. As shown in Fig. 3A the fragment generated

Fig. 3. The Man3-intermediate is phosphorylated via an ATP-dependent kinase. TLC-purified [3H]Man-labelled glycolipids III and IV were deaminated. The water-soluble fragments were analysed by Dionex-HPAEC (A). The fragment generated from glycolipid IV was analysed further on Bio-Gel P4 chromatography before (B) and after alkaline phosphatase treatment (C). The elution positions of the coinjected glucose oligomer standards are indicated at the top of (B). In (D) glycolipids were labelled in vitro using P. primaurelia membranes with GDP-[3H]Man in the presence of 10 mm ATPgS. The labelled glycolipids were processed and analysed as indicated in Fig. 1.

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P. primaurelia GPtdIns biosynthesis (Eur. J. Biochem. 267) 3389

Fig. 4. Glycolipid II contains an acid-labile modification linked to the mannosyl phosphate side chain. TLC-purified [3H]Man-labelled glycolipid II was reconsituted in liposomes. Liposomes containing glycolipid II were fused with P. primaurelia membranes. After a period of incubation glycolipids were extracted and analysed by TLC (B). (A) Standard incubation with GDP-[3H]Man. [3H]Man-labelled glycolipids II and III were TLC-purified then deaminated, subjected to mild acid hydrolysis and sized on Bio-Gel P4 column (C and D). The elution positions of the coinjected glucose oligomer standards are indicated at the top of each chromatogram. Glycolipid that migrates close to the front in (B) is due to a plate overloading.

phosphatase treatment. Compared with the intact fragment which eluted at 8 Gu (Fig. 3B) the fragment generated after alkaline phosphatase treatment eluted at 4.5 Gu (Fig. 3C), corresponding to the elution position of a Man3-intermediate [18]. The phosphorylation reaction is shown to be ATP dependent, as the addition of ATPgS, a nonhydrolysable analogue of ATP, during labelling with GDP-[3H]Man resulted in the accumulation of the Man3-intermediate (glycolipid V) (Fig. 3D). As the phosphate group is transferred only to the completed Man3-intermediate [7], the data suggest that the ATP-dependent mannosyl kinase involved in the phosphorylation step is specific for the Man3-intermediate. ATPgS has no effect on the biosynthesis of GPtdIns molecules in Toxoplasma gondii or Plasmodium as their GPtdIns types do not have a phosphate group linked to their core glycan (unpublished data). Glycolipid II is converted in vitro to the mannosyl phosphate containing glycolipid III To investigate a possible role for glycolipid II as a GPtdInsintermediate, we incorporated TLC-purified-[3H]Man-labelled

glycolipid II into liposomes. Liposomes containing glycolipid II were then incubated with membranes. The subsequent incubation resulted in the formation of glycolipid III (Fig. 4B). This result suggests that glycolipid II is a precursor of glycolipid III. We therefore investigated the structure of this glycolipid. We generated HNO2 fragments from glycolipid II and III and analysed them by Bio-Gel P4 column analysis. Although the neutral core glycans of these two glycolipids coeluted with Man3-AHM (Fig. 2D,E), their HNO2 fragments showed differences in their elution positions (Fig. 2A,B). The fragment generated from glycolipid II eluted at 12 Gu, 3.5 Gu more than the corresponding fragment generated from glycolipid III (8.5 Gu). Together, these results indicate that glycolipid II is more polar than glycolipid III, containing an acid-labile modification which is removed following dephosphorylation treatment with HF. Therefore, [3H]Man-labelled glycolipids II and III were TLC-purified, deaminated and treated with mild acid that cleaves the mannose belonging to the mannosyl phosphate side chain. As shown in Fig. 4, this treatment leads to the formation of a small fragment, which in the case of glycolipid III elutes at 1 Gu (Fig. 4C), corresponding to the elution position of a mannose standard

Table 1. Glycolipid II is localized in the lumen side of the ER. Glycolipids were labelled with GDP-[3H]Man in vitro using P. primaurelia membranes. After a period of incubation, an aliquot was subjected to PtdIns-PLC for 60 min at 4 8C. After PtdIns-PLC treatment and centrifugation, the glycolipids were extracted and analysed by TLC. Profiles of three separate experiments were integrated, before and after PtdIns-PLC treatment using the chroma software (Berthold LB 2842). The percentage of cleavage of each glycolipid is reported.

Lipid

Structure

Cleavage after PtdIns-PLC (%)

VII VI V IV III II I

Man1-GlcN-inositol-P-ceramide Man2-GlcN-inositol-P-ceramide Man3-GlcN-inositol-P-ceramide Man2-(P) Man-GlcN-inositol-P-ceramide Man2-(Man-P) Man-GlcN-inositol-P-ceramide Man2-(GalNAc-Man-P) Man-GlcN-inositol-P-ceramide EtN-P-Man2-(Man-P) Man-GlcN-inositol-P-ceramide

49 52 53 60 57 11 15

3390 N. Azzouz et al. (Eur. J. Biochem. 267)

and for glycolipid II at 3.5 Gu (Fig. 4D). This result indicates that glycolipid II is modified by an additional molecule or charge linked to the mannose of the mannosyl phosphate side chain.

q FEBS 2000

Glycolipid II contains GalNAc linked to the mannosyl phosphate side chain Glycolipid II was shown to lack a phosphoethanolamine group [7], which could explain the difference in size between the HNO2 fragments generated from glycolipids II and III. The difference of 2.5 Gu between the small fragments generated by mild acid treatment corresponds to the elution position of N-acetylhexosamine on the P4 column. The modification was indeed identified as an additional GalNAc linked to the mannosyl phosphate by labelling with UDP-N-acetyl-[3H] GalNAc. As shown in Fig. 5, in vitro labelling revealed that glycolipid II is the only glycolipid that can be labelled under these conditions (Fig. 5B). The transferred carbohydrate was shown to be GalNAc as determined by analysis of acidhydrolysed TLC-purified [3H]GalNAc-labelled glycolipid II. HPAEC monosacharide analysis showed (Fig. 5C) that the released labelled monosaccharide coeluted with a GalNH2 standard (resulting from the de-N-deacetylation of GalNAc during acid hydrolysis), confirming the nature of the modification in glycolipid II as GalNAc. Glycolipid II was generated following incubation with Dol-P-[3 H]Man reconstituted in liposomes (Fig. 1C); we suggest that Dol-P-Man is the donor of all mannose residues for P. primaurelia GPtdIns glycolipids. Both glycolipids I and II are localized at the luminal side of the ER The localization of the different GPtdIns intermediates within the ER membrane was investigated by their sensitivity to PtdIns-PLC. This method was used successfully in the case of T. brucei to study the topological localization of GPtdIns molecules [12,13]. Membranes were first incubated with GDP-[3H]Man to label the GPtdIns molecules. After 60 min incubation, an aliquot of membranes was treated with PtdInsPLC at 4 8C for 60 min in the absence of detergent. The supernatant containing hydrophilic moieties generated from glycolipids sensitive to PtdIns-PLC was discarded by centrifugation. Non-cleaved GPtdIns molecules were extracted by adding C/M/W (10 : 10 : 3, v/v/v) to the pellet. After TLC, the plates were analysed for radioactivity and the corresponding area of each glycolipid, before and after treatment with PtdIns-PLC, was integrated, and the percentage of cleavage was calculated. As summarized in Table 1, most of the GPtdIns molecules were sensitive to PtdIns-PLC (glycolipids III to VII)

Fig. 5. Glycolipid II contains GalNAc residue. Glycolipids were labelled either in vitro using P. primaurelia membranes with GDP[3H]Man (A) or UDP-[3H]GalNAc (B). The labelled glycolipids were extracted and processed as described in Fig. 1. TLC-purified [3H]GalNAclabelled glycolipid II was subjected to total hydrolysis followed by monosaccharide analysis on HPAEC (C). The positions of monosaccharide standards are indicated at the top of (C).

Fig. 6. Integrity of ER vesicles. Intactness of membranes was monitored by the accessibility of proteinase K to luminal ER BiP protein. P. primaurelia intact membranes (lane 1), membranes treated with PtdIns-PLC and proteinase K (50 mg´mL21; lane 2), membranes treated with PtdIns-PLC in the presence of 0.5% Triton X-100 and proteinase K (lane 3). BiP was detected by Western blot. Immunoreactive bands were visualized by adding a chemiluminescent peroxidase substrate.

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P. primaurelia GPtdIns biosynthesis (Eur. J. Biochem. 267) 3391

treatment, except glycolipids I and II. The sensitivity of glycolipids I and II to PtdIns-PLC was increased at 37 8C or when membranes were disrupted in the presence of 0.1% sodium deoxycholate at 37 8C (data not shown). Together, the data suggest that the final GPtdIns-anchor glycolipid I and glycolipid II are localized in the lumen of the ER membrane but not the early GPtdIns intermediates III±VII. The quality of the membranes was monitored before and after PtdIns-PLC treatment by determining the extent to which a luminal ER protein, BiP, was protected from the action of exogenously added proteinase K. We used T. brucei anti-BiP serum which was shown to cross react with T. gondii BiP homologue (unpublished results). As shown in Fig. 6, BiP was detected in intact membranes (Fig. 6, lane 1) as well as in membranes treated with PtdIns-PLC and proteinase K in the absence of detergent (Fig. 6, lane 2), indicating that PtdIns-PLC treatment has no effect on the integrity of the vesicles.

the donor of side chain mannose linked via phosphate to the GPtdIns core. Two potential donors were investigated: Dol-PMan, which is available on both sides of the ER, and GDP-Man which is available only on the cytoplasmic side of the ER. In this report we show that Dol-P-Man is not only the donor for the three mannoses belonging to the core glycan but is also the donor for the mannose linked to the phosphate group. Using Dol-P-[3H]mannose as the donor for mannose residues, we were able to label glycolipid II containing a mannosylphosphate side chain. Furthermore, we showed that the mannosyl phosphate modification is initiated by the phosphorylation of the Man3-intermediate via an ATP-dependent kinase. The structure of the phosphorylated intermediate was investigated by HPAEC chromatography, Bio-Gel P4 size exclusion chromatography and enzymatic/chemical treatments and was shown to have the following structure: Man-Man(P)Man-GlcNH2-inositol-phosphoceramide. We have also identified a hydrophilic intermediate (glycolipid II) containing GalNAc which is linked to the mannosyl phosphate modification. The hydrophilic fragment generated from glycolipid II was shown to be greater in size than the corresponding fragment generated from glycolipid III. Our previous work showed that glycolipid II does not contain an ethanolamine phosphate group, which explains its elution position on the P4 column. The data suggest an additional residue linked to the mannosyl phosphate side chain. Following metabolic labelling and structural analyses we identified GalNAc as an additional residue which is linked to the mannosyl phosphate. The fact that the final precursor (glycolipid I) does not contain GalNAc [7] indicates that the GalNAc residue is removed and that its presence is transitional. Furthermore we showed in vitro the conversion of glycolipid II to the mannosyl phosphate containing glycolipid III. Whether the removal of GalNAc and the generation of glycolipid III reflects in vivo processing is not known as the incubation of membranes with Dol-P-[3H]Man reconstituted in liposomes generated mainly

DISCUSSION We have used P. primaurelia as a model to study the biosynthesis of GPtdIns anchors in a cell-free system [7]. The sequence of GPtdIns biosynthesis in Paramecium, based on structural data, was shown to be the same as the pathways described for parasitic protozoa or mammalian cells. The biosynthesis starts with the transfer of GlcNAc to inositolphosphoceramide, followed by a transfer of three mannose residues, all derived from Dol-P-Man, and the addition of ethanolamine phosphate to the mannosylated core glycan [7]. The specificity of P. primaurelia is that the core glycan of polar GPtdIns intermediates is modified by a mannosyl phosphate unit linked via a phosphodiester bond to the mannose adjacent to the nonacetylated glucosamine. It has been shown that mannosylated GPtdIns-intermediates are synthesized in the cytoplasmic leaflet of the ER membrane [12,13]. For this reason experiments were performed to identify

Fig. 7. Biosynthetic pathway and hypothetical localization for P. primaurelia GPtdIns-glycolipids. This pathway is based on the structural characterization of different intermediates and their localization according to their accessibility to PtdIns-PLC. GalNAc-Man side chain addition occurs in two steps (w) or in block ( ). IPC, Inositol-phospho-ceramide.

3392 N. Azzouz et al. (Eur. J. Biochem. 267)

glycolipids II and IV but not glycolipid III. Our data on the topological localization of the final precursor and the GalNAccontaining glycolipid II intermediate indicates that they are not accessible to PtdIns-PLC, suggesting that they are exclusively localized on the luminal side of the ER membrane. Glycolipid III and the early GPtdIns intermediates, however, were shown to be present on the cytoplasmic side of the ER membrane. In T. brucei it was shown that most of the GPtdIns intermediates are synthesized at the cytoplasmic side of the ER [12,13]. These investigations in T. brucei suggest a translocation of at least one of the mature intermediates across the ER membrane bilayer to be transferred to proteins. We assume that the transient presence of the GalNAc-containing intermediate is necessary for the maturation of the GPtdIns molecules at the luminal side of the ER. We therefore suggest that the structure containing GalNAc is an intermediate recognized by an enzyme or enzyme complex to be translocated into the lumen of the ER. Once transferred to the lumen of the ER the GalNAc is removed, followed by the transfer of ethanolamine phosphate, leading to the formation of the final GPtdIns precursor which is transferred to protein. The oligosaccharide processing described leads us to propose the biosynthetic pathway and the hypothetical topological localization of different intermediates of P. primaurelia (Fig. 7). Further work is needed to check for the presence of other transiently glycosylated intermediates.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SFB 286), Fonds der Chemischen Industrie, Stiftung P. E. Kempkes and PROCOPE by DAAD/ARNT. We thank J. D. Bangs (University of Wisconsin, Madison, WI, USA) for antibodies and the T. brucei BiP homologue. The authors thank A. HuÈbel, M. Westermann and S. Becker for their critical reading of the manuscript.

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