Characterization of a Novel GDP-mannose:Serine-protein Mannose-1-phosphotransferase from Leishmania mexicana*

Jonathan M. MossDagger , Gavin E. Reid§, Kylie A. MullinDagger , Jody L. ZawadzkiDagger , Richard J. Simpson§, and Malcolm J. McConvilleDagger

From the Dagger  Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia and the § Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia

    ABSTRACT
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Abstract
Introduction
References

Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Manalpha 1-PO4-Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Manalpha 1-P to serine residues in a range of defined peptides. The presence and location of the Manalpha 1-PO4-Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn2+, utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO4 residues in the in vitro assays. Similarly, Man-PO4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.

    INTRODUCTION
Top
Abstract
Introduction
References

Parasitic protozoa of the genus Leishmania cause a spectrum of human and animal diseases that are transmitted by a sand fly vector. During their development in the digestive tract of the sand fly, these parasites differentiate from non-infective procyclic promastigotes to infective metacyclic promastigotes that target mammalian macrophages when introduced into the host during the insect's blood meal. Following their internalization into the macrophage phagolysosome, metacyclic promastigotes differentiate into a replicative amastigote stage and eventually rupture the host cell and perpetuate disease by infecting other host cells. A number of cell-surface and secreted virulence factors are thought to be crucial for the survival of these different developmental stages in their respective host environments. These include an abundant glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG)1 (1-3) and a number of secreted glycoproteins and proteophosphoglycans (PPGs) (reviewed in Refs. 4 and 5). Strikingly, both the cell-surface LPGs and the secreted molecules are elaborated with structurally related phosphoglycan chains that are thought to be the major functional determinants of these molecules.

The secreted glycoproteins and proteoglycans of Leishmania have been shown to form distinct macromolecular complexes in the flagellar pocket and extracellular milieu (4). The most intensively characterized of these molecules are the secreted acid phosphatases (sAPs), which aggregate into large pearl-like filamentous polymers (6, 7). Early studies indicated that sAPs were heavily glycosylated and phosphorylated enzymes that contained glycan epitopes characteristic of LPGs (6, 8, 9). We have recently shown that Leishmania mexicana sAP is extensively modified with Manalpha 1-PO4 residues that are linked to serine residues in the polypeptide backbone (10). This unusual type of linkage, in which a monosaccharide is linked to protein via a phosphodiester bridge, has been termed phosphoglycosylation (11) and may be widespread in several lower eukaryotes (12). In L. mexicana, the Manalpha 1-PO4 residues can be further elaborated with alpha 1-2-linked mannose oligosaccharides or short chains of phosphorylated di- or trisaccharides (10), which are also found in the long phosphoglycan chains (as capping structures or internal repeat units, respectively) of L. mexicana LPG (see Fig. 1). Interestingly, the nature of these modifications may be influenced by the size of the Ser/Thr-rich repeat domains in the polypeptide backbone. For example, sAP-1 contains a relatively short Ser/Thr-rich domain and is modified primarily with mannose oligomers, whereas sAP-2, which contains a longer stretch of Ser/Thr-rich repeat sequences, is extensively modified with short phosphoglycan chains (7, 10). In addition to sAP, two heavily phosphoglycosylated PPGs have been recently characterized from leishmanial promastigotes and amastigotes (13-16). Promastigote PPGs are produced by all species of Leishmania and form a network of unbranched filaments in the center of aggregated promastigotes (14, 16). Amastigote PPGs (aPPGs) are produced by most species and accumulate to very high levels in the lumen of the parasitophorous vacuole and in the extracellular space of lesions (13, 15). The polypeptide backbones of PPGs differ from sAPs in being rich in proline and alanine as well as serine and threonine and are more extensively modified with phosphoglycan chains, which may account for as much as 90% of the molecule (13-15). Whereas the phosphoglycan chains of Leishmania major promastigote PPG are similar to those of the corresponding LPG, L. mexicana aPPGs are elaborated with novel stage-specific phosphoglycans that are distinct from those found on both promastigote sAPs and LPGs (15). However, all these glycans are thought to be linked exclusively to the polypeptide backbone via a Manalpha 1-PO4-Ser core sequence (14, 15).

Recent functional studies have shown that these molecules may be important for parasite infectivity and survival in both the insect and the mammalian host. L. mexicana aPPG is a potent activator of the complement cascade via the mannan-binding lectin pathway and may effectively deplete the local supply of complement components needed for parasite lysis (17). The release of PPGs into the phagolysosome may also inhibit membrane fusion events and contribute to the formation of large parasitophorous vacuoles, which are characteristic of New World species such as L. mexicana (18). On the other hand, secretion of sAP and promastigote PPG filamentous networks by the promastigote stages may be responsible for the tendency of cells to aggregate into large clusters in stationary growth. Cell aggregation may improve the efficiency of transmission during the sand fly bite or afford cells within the cluster some protection from complement lysis during the early stages of infection (4).

As this type of protein glycosylation is unique to Leishmania, the enzymes involved in initiating and assembling these glycans may be potential targets for new anti-leishmanial drugs. At present, several enzyme activities have been detected in cell-free assays and detergent extracts that are involved in the synthesis of the phosphoglycan chains of LPG, but that may also be involved in the synthesis of the shorter phosphoglycan chains of sAP and PPGs. These include two elongating enzymes, a putative alpha -mannose-1-phosphotransferase (MPT) and a beta 1-4-galactosyltransferase, that transfer Manalpha 1-PO4 and Gal from GDP-Man and UDP-Gal, respectively, to form the repeating Galbeta 1-4Manalpha 1-PO4 backbone of both the LPG and PPG glycans (Fig. 1) (19, 20). In addition, a beta 1-3-galactosyltransferase activity that adds the L. major-specific side chains to these repeat units has also been characterized (21). Interestingly, a separate MPT activity is thought to be involved in adding the first Man-PO4 residue to the LPG anchor precursor and thus to be required to initiate LPG phosphoglycan biosynthesis (22). As one of the enzymes involved in synthesizing the LPG anchor is localized to the Golgi apparatus (23), it is likely that both the initiating MPT and phosphoglycan chain-elongating and -branching enzymes are also localized in either a Golgi or post-Golgi compartment. This is consistent with the finding that a GDP-Man transporter required by these MPTs is also localized to the Golgi apparatus (24) and that phosphoglycan chain elongation is inhibited by the Golgi-perturbing ionophore, monensin (25).


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Fig. 1.   Structure of the phosphoglycan chains of L. mexicana promastigote LPG and sAP. The glycosylphosphatidylinositol (GPI) anchor of LPG is modified with a single phosphoglycan chain comprising a long domain of phosphodi- and phosphotrisaccharide repeat units and a mannose oligosaccharide cap at the nonreducing terminus (2, 3). In contrast, a Ser/Thr-rich domain in the secreted sAP glycoproteins is modified either with mannose oligosaccharides or short phosphoglycan chains that are capped with mannose oligosaccharides. Both classes of glycans are thought to be attached to serine residues via a Manalpha 1-PO4 linkage (10). The more complex branched phosphoglycans of the L. mexicana aPPGs are not shown (15).

In this study, we have identified a peptide-specific MPT (pep-MPT) activity from L. mexicana promastigotes that is most likely involved in initiating protein phosphoglycosylation. Mass spectrometry of the glycopeptides containing the Man-PO4 modification provides the first direct characterization of this novel linkage. We show that pep-MPT is most active against peptides containing the Ser/Thr-rich sequences of endogenous polypeptide acceptors and that it is not inhibited by glycan acceptors of the LPG phosphoglycan-initiating and -elongating MPTs. Furthermore, we provide evidence that this enzyme occurs in a distinct subcompartment of the Golgi apparatus from enzymes involved in phosphoglycan chain elongation.

    EXPERIMENTAL PROCEDURES

Materials-- Alkaline phosphatase, GDP-Man, UDP-Gal, and stachyose were from Sigma; jack bean alpha -mannosidase was from Boehringer Mannheim. The synthetic oligosaccharide acceptor L2 (Galbeta 1-4Manalpha 1-PO4-(CH2)8CH=CH2) was generously provided by Professor Michael Ferguson (University of Dundee). GDP-[3H]Man was prepared using previously described methods (26, 27). Briefly, [2-3H]Man-6-PO4 was synthesized enzymatically from [2-3H]Man (NEN Life Science Products) using yeast hexose kinase and then converted to the sugar nucleotide using a mixture of yeast proteins supplemented with snake venom pyrophosphatase and glucose-1,6-diphosphate. The yeast proteins, corresponding to a 50-70% ammonium sulfate cut, were prepared from protease A-deficient yeast strain SC295. GDP-[3H]Man was purified on a column (1 ml) of concanavalin A-Sepharose (Amersham Pharmacia Biotech) that was washed with 15 mM ammonium acetate, 1 mM MgCl2, and 1 mM CaCl2 and then eluted with the same buffer containing 50 mM alpha -methylmannoside. Fractions containing radioactivity were desalted on a column of Sephadex G-10 eluted in water. Polyclonal antibodies against L. major Rab1 protein (28) and Trypanosoma brucei BiP (29) were generously provided by Dr. Emanuela Handman (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) and Dr. James Bangs (Department of Biochemistry, University of Wisconsin, Madison, WI), respectively.

Synthesis of Peptide Substrates-- Peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a Applied Biosystems Model 431A peptide synthesizer. Peptides were cleaved from the resin using 95% trifluoroacetic acid (3 h, 25 °C), recovered by extraction with diethyl ether, and purified on a C18 reversed-phase column (4.6, inner diameter, × 100 mm; Brownlee) eluted with acetonitrile (0-60%) in 0.1 M ammonium acetate over 20 min at a flow rate of 1 ml/min. Reductive methylation of sapp-1 was carried out in 0.2 M borate buffer, pH 9.0 (140 µl), containing 16 mM formaldehyde and 0.1 mM NaB3H4 (5 mCi) at 0 °C for 10 min (30). The mixture was acidified with 1 M acetic acid to remove excess NaB3H4, and the radiolabeled peptides were recovered after application to a Sep-Pak C18 cartridge (Waters) following elution with 40% acetonitrile.

Preparation of Parasite Membranes-- Promastigotes of L. mexicana (strain MNYC/BZ/62/M379) were cultivated at 27 °C in RPMI 1640 medium supplemented with 10% fetal bovine serum and harvested at mid-log growth unless otherwise stated. Amastigotes were isolated from tail lesions in BALB/c mice as described by Bates et al. (31). Cells were harvested by centrifugation (750 × g, 10 min, 27 °C); washed once with phosphate-buffered saline; repelleted; and suspended in 50 mM HEPES-NaOH, pH 7.4, containing 50 mM KCl, 10 mM MgCl2, 10 mM MnCl2, 5 mM Na2EGTA, 2 mM dithiothreitol, 1 mM ATP, 0.1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, and 0.2 mM Nalpha -p-tosyl-L-lysine chloromethyl ketone (buffer A). Cell disruption was carried out by probe sonication (MSE Soniprep 150 Ultrasonic Disintegrator) at 0 °C using 8 × 10-s bursts at an amplitude of 24 µ with 20-s intervals between each burst. Greater than 90% cell lysis was confirmed by microscopy. Intact cells and large cell fragments were removed by centrifugation (750 × g, 5 min, 4 °C), and microsomal membranes in the supernatant were recovered by ultracentrifugation at 100,000 × g (30 min, 4 °C). The pelleted crude microsomal membranes were resuspended in buffer A or in buffer A containing 1% Triton X-100 and either used immediately or stored frozen at -70 °C. Detergent-extracted material (10 min, 0 °C) was centrifuged at 14,000 × g (5 min, 4 °C) to remove insoluble material. Triton X-100 proved to be the most effective of several detergents tested for recovery of pep-MPT activity; these included Nonidet P-40, CHAPS, Zwittergent 3-12, nonanoyl-N-methylglucamide (MEGA-9), and n-octyl beta -D-glucopyranoside. Protein was precipitated in chloroform/methanol/water (32) prior to quantitation using the BCA assay (Pierce).

Mannose-1-phosphotransferase Assays-- pep-MPT activity was measured using two assays. In the first method, microsomal membranes (2 × 107 cell eq, 20-60 µg of protein) were resuspended in buffer A (50 µl) containing GDP-[3H]Man (160,000 cpm, 0.1-0.5 mM) and 0.4 mM synthetic peptide and incubated at 32 °C for 30 min. Triton X-100-solubilized membranes were assayed under identical conditions, except that the reaction mixture contained 0.1% Triton X-100 and assays were performed at 16 °C. The reaction mixture was diluted with 0.1 M ammonium acetate (950 µl), and the enzyme reaction was terminated by boiling (3 min). Precipitated proteins was removed by centrifugation (14,000 × g, 5 min), and the supernatant was loaded onto Sep-Pak C18 cartridges pre-equilibrated in 0.1 M ammonium acetate. After washing the cartridge with 0.1 M ammonium acetate (10 ml) to remove unincorporated GDP-[3H]Man, [3H]Man-labeled peptide was eluted with 40% acetonitrile (3 ml), and eluted radioactivity was measured by scintillation counting.

In the second method, Triton X-100-solubilized membranes (2 × 107 cell eq, 20-60 µg of protein) were diluted in buffer A (15 µl) containing 0.1% Triton X-100, GDP-Man (0.5 mM), and 3H-labeled peptide (500,000 cpm, 0.16 mM). Assay mixtures were incubated at 16 °C for 15 min. In the substrate competition experiments described in the legend to Fig. 6, the reaction mixtures contained unlabeled peptide or glycan acceptors at 0-5 mM final concentration. Reactions were terminated by boiling (3 min), and the precipitated protein was removed by centrifugation at 14,000 × g for 5 min. Underivatized and mannosylated 3H-labeled peptides in the reaction mixture were resolved from each other by analytical thin-layer electrophoresis of 4 µl of the reaction mixture. Alternatively, 3H-labeled peptides were recovered on Sep-Pak C18 cartridges as described above for subsequent chemical and enzymatic analysis.

HPLC and HPTLC Analyses-- Underivatized and Man-PO4-modified peptides were separated on a C18 reversed-phase HPLC (RP-HPLC) column (4.6, inner diameter, × 100 mm; Brownlee) eluted with 0-35% acetonitrile in 0.1 M ammonium acetate at 1 ml/min over 30 min. Peptides were detected by UV absorbance at 280 nm (for sapp 1, 2, 4-6) or 216 nm (for Ser11 and sapp-3). Radioactivity in 0.2-ml fractions was determined by scintillation counting. Man-PO4-modified peptide and lipo-oligosaccharide L2 acceptors were resolved on aluminum-backed Silica Gel 60 HPTLC sheets (Merck) developed in chloroform, methanol, and 0.25% KCl (10:10:3, v/v) over 10 cm. HPTLC sheets were sprayed with EN3HANCE (NEN Life Science Products) and exposed to Biomax MR film (Eastman Kodak Co.) at -70 °C.

Thin-layer Electrophoresis-- Man-PO4-modified 3H-labeled peptides were resolved from unmodified 3H-labeled peptides by thin-layer electrophoresis on plastic-backed cellulose thin-layer sheets (20 × 20 cm; Macherey Nagel). Aliquots of the reaction mixture (containing 100,000 cpm) were spotted onto the cellulose sheets, which were then saturated with pyridine/acetic acid/water (1:10:89, v/v) and subjected to electrophoresis (500 V, 30 mA, 75 min) under a layer of petroleum spirit and pyridine/acetic acid/water (1:10:89, v/v) in each electrophoresis chamber. Radioactivity on the dried cellulose sheets was detected using an automatic TLC scanner (Berthold) and/or by fluorography as described above. Incorporation of radioactivity into phosphoglycosylated peptides was quantitated by scraping the cellulose containing the bands using the fluorograph as template, extracting the cellulose with 30% acetonitrile (2 × 250 µl), and liquid scintillation counting of the extracts.

Enzyme and Chemical Analyses of Phosphoglycosylated Peptides-- Phosphoglycosylated peptides were digested with jack bean alpha -mannosidase (30 µl, 50 units/ml) in 0.1 M sodium acetate buffer, pH 5.0 (18 h, 37 °C), with or without subsequent digestion in alkaline phosphatase (2000 units/ml) in ammonium bicarbonate, pH 8.5 (7 h, 37 °C). For recovery of 3H-labeled glycans, the enzyme digests were desalted on a column (400 µl) of AG 50-X12 (H+) over AG 3-X8 (OH-). Alternatively, 3H-labeled peptides were recovered on a Sep-Pak C18 cartridge (as described above), which was eluted with 40% acetonitrile. Specific cleavage of the Manalpha 1-PO4 bond was achieved with mild acid hydrolysis in 40 mM trifluoroacetic acid (12 min, 100 °C) (3). The acid was removed by drying under high vacuum in a Speed-Vac concentrator. 3H-Labeled peptides were analyzed by thin-layer electrophoresis as described above, whereas 3H-labeled glycans were analyzed by HPTLC using 1-propanol/acetone/water (9:6:5, v/v) as the solvent system (33).

Electrospray Ionization Mass Spectrometry-- Unmodified and phosphoglycosylated peptides were analyzed with a triple quadrupole mass spectrometer (Finnigan MAT Model TSQ-700) equipped with a Finnigan MAT electrospray ionization (ESI) source and rapid capillary RP-HPLC. The column used in this study (0.2, inner diameter, × 150 mm; Vydac C18) was fabricated and operated as described elsewhere (34). The column was developed with a linear 30-min gradient at 1.6 µl/min from 0 to 100% solvent B, where solvent A was 0.1% trifluoroacetic acid and solvent B was 60% acetonitrile containing 0.1% trifluoroacetic acid (35). The ESI needle was operated at -4.5 kV. The sheath liquid was methoxyethanol delivered at 3 µl/min. Nitrogen sheath and auxiliary gasses were supplied at 30 p.s.i. and 15 units (arbitrary units), respectively. The heated capillary was set at 150 °C. Mass spectra were collected every 3 s in centroid mode. Peptide masses were calculated using Finnigan MAT BIOMASSTM software. For tandem mass spectrometric peptide sequence analysis, quadrupole Q1 was operated with a resolution of 2-2.5 Da and Q3 with a resolution of 1-1.5 Da (35). The collision cell offset voltage was calculated by multiplying the mass of the ion selected for collision-induced dissociation by 0.04. The daughter ion offset voltage was set at twice the collision cell offset value. The parent ion offset voltage was set at one-third the value of the daughter ion offset voltage. Argon was used as the collision gas at a pressure of 2-2.5 millitorr.

Characterization of Endogenous Polypeptide and LPG Acceptors-- Sonicated microsomal membranes were incubated with GDP-[3H]Man in the presence of exogenous peptide acceptors and UDP-Gal as described above. Membranes were recovered by centrifugation (14,000 × g, 5 min), washed with water to remove residual peptide, and recentrifuged, and the supernatants were combined for recovery of the peptide on a Sep-Pak column as described previously. The membrane pellet was subsequently extracted twice in 1% Triton X-100 (200 µl) for 10 min at 30 °C to remove LPG and soluble phosphoglycan and proteoglycans. Insoluble material was recovered by centrifugation; the supernatants were combined and subjected to two-phase partitioning with 1-butanol; and the detergent-free aqueous phase was dried in a Speed-Vac concentrator. This material was resuspended in 0.1 M NH4OAc containing 5% 1-propanol and loaded onto a minicolumn of octyl-Sepharose equilibrated in the same buffer. The column was washed with 0.1 M NH4OAc containing 5% 1-propanol and then with 40% 1-propanol to elute bound LPG (36). The Sep-Pak-eluted peptide, octyl-Sepharose-purified LPG, and Triton X-100-insoluble material were hydrolyzed in 40 mM trifluoroacetic acid (12 min, 100 °C), and the acid was removed under reduced pressure in a Speed-Vac concentrator (36). Acid-released glycans were dephosphorylated with alkaline phosphatase and desalted by passage over a column of AG 50-X12 (H+) over AG 3-X8 (OH-) for analysis by HPTLC (3).

Subcellular Fractionation-- L. mexicana promastigotes (8 × 108) were harvested by centrifugation (750 × g, 10 min), washed with phosphate-buffered saline, and then resuspended (2 × 108 cells/ml) in assay buffer A supplemented with 0.25 M sucrose. The cell suspension was transferred to a prechilled nitrogen cavitation bomb (Kontes Glass Co.) equilibrated at 450 p.s.i. N2 pressure for 15 min at 0 °C, and cells were lysed by abruptly releasing the pressure. Equilibration and expulsion of the suspension were repeated twice more to achieve complete cell lysis. Cell debris and nuclei were removed by centrifugation of the lysate at 3000 × g for 10 min, and the supernatant was layered on top of a linear sucrose gradient. The sucrose gradient (density = 1.05-1.27 g/ml) was prepared by layering 10 0.8-ml fractions (0.25-2 M sucrose in 0.25 mM HEPES-NaOH, pH 7.4) over a sucrose cushion (2.5 M) in Ultraclear centrifugation tubes (Beckman Instruments) and centrifuging at 218,000 × g for 1 h at 4 °C in a Beckman L-80 ultracentrifuge using an SW 41 rotor. Organelles in the cell lysate were fractionated by equilibrium centrifugation at 218,000 × g for 6 h at 4 °C. Fractions (0.5 ml) were collected from the bottom of the tube, and densities were calculated by measuring refractive index. The distribution of leishmanial BiP and Rab1, markers for the bulk endoplasmic reticulum and cis-Golgi cisternae, respectively (28, 29), were determined by immunoblotting. Proteins in each fraction were precipitated in biphasic mixtures of chloroform/methanol/water (32), measured by the BCA assay, separated by SDS-polyacrylamide (12%) gel electrophoresis, and transferred to nitrocellulose by electroblotting. Strips of nitrocellulose corresponding to regions containing the BiP (70 kDa) and Rab1 (25 kDa) proteins were cut and processed individually. For detection of BiP, the nitrocellulose was blocked with 0.05% Tween 20 in Tris-buffered saline, incubated with rabbit anti-BiP antibody (1 h), washed with 0.05% Tween 20 in Tris-buffered saline, and then probed with horseradish peroxidase-conjugated anti-rabbit antibody (1:10,000 dilution; Silenus Laboratories, Pty., Ltd.) in 0.05% Tween 20 in Tris-buffered saline containing 1% powdered skim milk (1 h) and visualized with the ECL Western detection system (Amersham Pharmacia Biotech). Nitrocellulose strips containing Rab1 protein were probed in the same way, except that the nitrocellulose was initially blocked with 0.05% Tween 20 in Tris-buffered saline with 1% powdered skim milk, the rabbit anti-L. major Rab1 antibody was used at 1:500 dilution, and incubation with the secondary antibody was done in 0.05% Tween 20 in Tris-buffered saline.

    RESULTS

Phosphoglycosylation of Peptides Containing the Ser/Thr-rich Repeat Sequences of L. mexicana sAP-- We have previously shown that the major secretory proteins of L. mexicana promastigotes are modified with phosphoglycan chains that are probably linked to the polypeptide backbone via a Manalpha 1-PO4-Ser linkage (10). To confirm this linkage by mass spectrometry and to develop an assay for the initiating enzyme, a series of peptides containing the Ser/Thr-rich peptide repeats from L. mexicana sAP-1 and sAP-2 (7) were synthesized (sapp-1, -2, -3, and -4 in Table I) and incubated with L. mexicana promastigote membranes and GDP-[3H]Man as a sugar donor. [3H]Man-labeled peptides were purified on a Sep-Pak C18 cartridge and further analyzed by RP-HPLC. As shown in Fig. 2, incubation of the membranes with sapp-1 (containing two Ser-rich motifs) generated at least five 3H-labeled peaks that eluted earlier than the unmodified peptide on RP-HPLC. These peaks were not detected when membranes were incubated in the absence of either the exogenous peptide or GDP-Man (data not shown). Treatment of the total peptide mixture or the individual HPLC-purified peaks with either jack bean alpha -mannosidase (data not shown) or mild trifluoroacetic acid (to selectively cleave hexose 1-phosphate linkages) generated a single labeled component in each case that comigrated with mannose on HPTLC (Fig. 2C). These data suggested that sapp-1 was being variably modified with one or more Manalpha 1-PO4 residues. This was confirmed by positive ion ESI-MS of the HPLC-purified phosphomannosylated peaks 1 and 2 in Fig. 2. Multiply charged ions were obtained that were in agreement with the calculated mass for sapp-1 modified with one or two hexose phosphate residues, respectively (Table II). As the ionization of these phosphomannosylated peptides was relatively poor, a smaller peptide (sapp-2) containing a single Ser/Thr-rich repeat unit and a C-terminal lysine residue was used as substrate. As with sapp-1, several [3H]Man-labeled peaks were generated when this peptide was incubated with sonicated membranes (Fig. 3A). Positive ion ESI-MS of the two major peaks contained both [M + H]+ and [M + 2H]2+ ions corresponding to sapp-2 with either one (Fig. 3B) or two (Fig. 3C) hexose-PO4 residues (Table II). Fragment ions corresponding to the loss of one or two hexose residues were also evident in these mass spectra, indicating that the Man-PO4 linkage is relatively labile under the ionization conditions employed. Taken together, these data indicate that peptides containing the same Ser/Thr-rich domains as endogenous polypeptides are modified with one or more single Manalpha 1-PO4 residues, providing the first evidence for the presence of a pep-MPT activity.

                              
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Table I
Series of acceptor substrate peptides synthesized containing 0-2 serine-rich repeats


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Fig. 2.   RP-HPLC analysis of phosphomannosylated peptides synthesized in vitro. Following incubation of promastigote microsomal membranes with sapp-1 and GDP-[3H]Man, unmodified and phosphomannosylated peptides were purified on a Sep-Pak C18 cartridge and resolved by RP-HPLC employing a gradient of acetonitrile (0-35%) in 0.1 M ammonium acetate. Peptides were detected by UV absorbance at 280 nm (A) and by scintillation counting for [3H]Man-labeled peptide (B). Fractions containing peaks 1-5 were subjected to mild acid hydrolysis, and acid-labile (phosphodiester-linked) glycans were analyzed by HPTLC (C). Only the region of the HPTLC in which monosaccharides (Glc, Gal, and Man) and disaccharides (i.e. Galbeta 1-4Man) migrated (RF = 0.3-0.8) is shown. No bands were observed in other regions of the HPTLC.

                              
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Table II
ESI-MS of RP-HPLC-separated unmodified and phosphomannosylated peptides after incubation with L. mexicana promastigote membranes and GDP-[3H]Man


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Fig. 3.   Positive ion ESI-MS mass spectra of mono- and diphosphomannosylated sapp-2 peptides. A, RP-HPLC profile (A280) of unmodified and phosphomannosylated sapp-2 peptides; B and C, positive ion ESI-MS of monophosphomannosylated and diphosphomannosylated peaks, respectively. Several minor ions in the spectrum shown in B (m/z 1798.9, 1637.2, and 900.2) were derived from a contaminating peptide with the same sequence as sapp-2 minus one of the glycine residues.

Collision-induced dissociation of the monophosphomannosylated sapp-1 and sapp-2 peptides provided negligible or incomplete sequence information on the site(s) of Man-PO4 substitution, reflecting the relatively poor ionization of these peptides. However, complete sequence information was obtained for monophosphomannosylated sapp-3, a peptide with a single serine-rich motif and two C-terminal lysine residues (Tables I and II). Tandem mass spectrometry of the monosubstituted sapp-3 [M + 2H]2+ ion (m/z 995.4) resulted in the loss of mannose and fragmentation along the peptide backbone to produce a series of N-terminal (b-type) and C-terminal (y-type) ions (Fig. 4, A and B). The y series of fragment ions were particularly informative. As shown in Fig. 4, the y1-8 fragment ions observed in the mass spectrum corresponded to the unmodified peptide, indicating that none of the C-terminal residues in the peptide, including Ser15 and Ser16, were phosphorylated. In contrast, two series of y9-11 ions, differing in mass by 80 Da (corresponding to both unmodified and phosphorylated forms of Ser8-10), and one series of y12-16 ions corresponding to the phosphorylated peptide were observed in the mass spectrum (Fig. 4, A and B). Additional fragment ions corresponding to loss of H3PO4 from the phosphorylated peptides, diagnostic ions for serine- and threonine-phosphorylated peptides (37), were also present (yn' in Fig. 4, A and B). The progressive decrease in ion intensity of the y9-12 ions for the unmodified peptide and the corresponding increase in ion intensity of the y9-12 ions for the phosphorylated peptide (Fig. 4C) suggest that all four Ser residues in the central domain of this peptide were partially phosphorylated. Whereas the b series of fragment ions was less complete, the identification of the expected b2-5 series for the unmodified peptide indicates that neither Ser1 nor Ser2 is phosphorylated. These data indicate that sapp-3 is heterogeneously modified with Manalpha 1-PO4 on Ser7-10 and also suggest that none of the Thr residues are modified.


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Fig. 4.   Positive ion collision-induced dissociation mass spectrum of monophosphomannosylated sapp-3. Shown is the collision-induced dissociation mass spectrum of the monophosphomannosylated sapp-3 peptide [M + 2H]2+ ion at m/z 995.4. Shown in A are the predicted masses for b-type fragment ions (top level) and y-type fragment ions (bottom level) derived from either the unmodified peptides (yn) or the phosphorylated peptides (yn* for yn+80 and yn' resulting from loss of H3PO4 from the yn* peptide ion). Ions observed in the mass spectrum in B are underlined. Because the mannose residue was readily lost during collision-induced dissociation, only the phosphorylated form of the modified peptide was observed. The relative intensities of the y9-12 and y9-12+80 ions (as a percent of the sum of the two ions) are indicated in C.

Properties of the pep-MPT Activity-- pep-MPT activity in detergent-solubilized microsomes was not affected by inclusion of amphomycin (0.6 mg/ml), a potent inhibitor of dolichol-phosphate mannose synthesis,2 indicating that the mannose phosphate residue was being directly transferred from GDP-Man (data not shown). Although pep-MPT activity was solubilized from microsomal membranes (>80%) with a range of non-ionic detergents (see "Experimental Procedures"), Triton X-100 was the most effective at retaining enzyme activity (Fig. 5A) (data not shown). However, enzyme activity was considerably more labile after detergent solubilization, as maximal activity occurred at 16 °C, rather than at 32 °C, when non-solubilized membranes were assayed (data not shown). The detergent-solubilized pep-MPT activity displayed a pH optimum at 7.4 (Fig. 5B) and had an apparent Km for GDP-Man of 180 µM (Fig. 5C). All activity was abolished when 5 mM EGTA was included in the reaction mixture in the absence of added divalent cations, but was restored to maximal levels with the addition of 10 mM MnCl2 (i.e. 5 mM free Mn2+) (Fig. 5D). MgCl2 also stimulated activity, but was not as effective as MnCl2 and did not significantly further stimulate the effect of adding MnCl2 alone (Fig. 5D). Addition of 5 mM free Ca2+ had no effect on the pep-MPT activity in the absence (Fig. 5D) or presence (data not shown) of MnCl2 or MgCl2. Interestingly, the pep-MPT activity varied markedly in a growth- and stage-dependent manner (Fig. 5A). Rapidly growing (procyclic) promastigotes contained 5- or 10-fold higher activity than either late stationary (metacyclic) promastigotes or freshly isolated lesion amastigotes, respectively.


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Fig. 5.   Properties of L. mexicana pep-MPT. A, levels of pep-MPT activity in promastigotes from early logarithmic (L) and late stationary (S) phase cultures and from lesion-derived amastigotes (A). Specific activity was measured in sonicated membranes with or without solubilization in 1% Triton X-100 (TX-100). Total pep-MPT activity in the detergent extracts was ~80% of that in the sonicated membranes in the absence of detergent. B-D, the effect of pH, GDP-Man concentration, and various cations, respectively, on pep-MPT activity. The standard assay buffer was used in all experiments, except for the cation dependence experiment, where buffer A minus MgCl2 and MnCl2 was used and cations were supplemented as indicated.

The pep-MPT Activity Is Distinct from the LPG Phosphoglycan-initiating and -elongating MPTs-- Although pep-MPT utilizes exogenously added peptides, it was possible that this activity was due to two previously characterized leishmanial MPT activities that are thought to be required for initiation and elongation of the LPG chain biosynthesis (20, 22). These oligosaccharide MPT activities add Man-PO4 to terminal galactose residues on either the LPG anchor precursor or phosphodisaccharide repeat units on the growing LPG phosphoglycan chain, respectively (20, 22). A second assay was developed to assess whether synthetic oligosaccharide acceptors for these MPTs competed with the peptide substrates for pep-MPT activity. In this assay, N-terminally 3H-labeled sapp-1 peptide (at a concentration of 2 × Km) and unlabeled GDP-Man were added to microsomal membranes in the absence or presence of unlabeled oligosaccharide or peptide substrates. In the absence of competitive substrate, ~30% of the 3H-labeled sapp-1 peptide was modified with Man-PO4 residues over a 30-min incubation period, as shown by the appearance of additional labeled bands on thin-layer electrophoresis that migrated more rapidly toward the cathode (Fig. 6A). The additional bands corresponded to the sapp-1 peptide with one, two, or three Manalpha 1-PO4 residues, as sequential treatment of the peptide mixture with jack bean alpha -mannosidase and alkaline phosphatase collapsed these bands back to a single labeled species that comigrated with unmodified sapp-1 (Fig. 6B). As expected, the addition of 1 mM unlabeled sapp-1 resulted in a 55% decrease in the extent to which the reductively methylated 3H-labeled sapp-1 peptide was modified (Fig. 6C). In contrast, the oligosaccharide acceptors for the LPG-specific MPTs, L2 (Galbeta 1-4Manalpha 1-PO4-(CH2)8CH=CH2) and stachyose (Galalpha 1-6Galalpha 1-6Glcalpha 1-2Fru), did not inhibit the pep-MPT activity when present at a 6-30-fold excess over the concentration of the sapp-1 peptide (Fig. 6C). The concentration of L2 used in these experiments was higher than that used previously (~600 µM) to achieve close to maximal saturation of the putative elongating MPT (20). Moreover, in separate experiments, unlabeled sapp-4 was found not to inhibit the phosphomannosylation of L2. As shown in Fig. 7, incubation of detergent-solubilized microsomal membranes with L2 (1 mM) and GDP-[3H]Man generated a labeled product with a slower HPTLC mobility compared with unmodified L2 (compare lanes 1 and 3). This product has previously been shown to correspond to L2 modified with a single terminal Man-1-PO4 residue (20). Significantly, the modification of L2 by the putative elongating MPT was not affected by the inclusion of sapp-4 (up to 0.5 mM) in the assay (Fig. 7, compare lanes 1 and 2). As the apparent Km of pep-MPT for sapp-4 is 0.05 mM (Table III), these data support the notion that the elongating MPT and pep-MPT are separate enzyme activities. Similar experiments were not performed with substrates of the initiating MPT (stachyose or LPG anchor) because high concentrations of GDP-Man (1 mM) (22) are needed to detect this activity, precluding the use of GDP-[3H]Man.


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Fig. 6.   Substrate specificity of pep-MPT. The substrate specificity of pep-MPT was tested using a competition assay. Triton X-100-solubilized microsomal membranes were incubated with 3H-labeled sapp-1 and GDP-Man in the absence or presence of the indicated competitive substrate (1 mM), and the labeled products were analyzed by thin-layer electrophoresis. A, when products were analyzed after a 30-min incubation (30') in the absence of competitive substrate, several additional labeled bands (not present in the zero (0') time point) were generated that migrated toward the cathode. B, sequential digestion of the products in the 30-min time point with jack bean alpha -mannosidase (JBAM) and alkaline phosphatase (AP) collapsed these bands back to unmodified sapp-1, indicating that these bands were differentially modified with Manalpha 1-PO4. C, inhibition of 3H-labeled sapp-1 phosphomannosylation by glycan acceptors of the LPG-specific MPTs (L2 and stachyose) and various unlabeled peptide substrates (sapp-1, sapp-4, sapp-5, sapp-6, and Ser11) is shown using standard incubation conditions. The structures of L2 and stachyose are Galbeta 1-4Manalpha 1-PO4-(CH2)8CH=CH2 and Galalpha 1-6Galalpha 1-6Glcbeta 1-2Fru, respectively. The mean values of three experiments are shown. O indicates the sample loading origin on the thin-layer electrophoresis sheets.


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Fig. 7.   sapp-4 does not inhibit phosphomannosylation of L2. Microsomal membranes were solubilized in 1% Triton X-100 and incubated with the synthetic lipo-oligosaccharide acceptor L2 (1 mM) (lane 1) or both L2 (1 mM) and sapp-4 (0.5 mM) (lane 2) or sapp-4 (0.5 mM) (lane 3), together with GDP-[3H]Man (0.5 mM). After incubation (30 min, 16 °C), the reaction was stopped, and the labeled lipo-oligosaccharide and peptide acceptors were recovered on a Sep-Pak C18 cartridge (20) and analyzed by HPTLC in chloroform, methanol, and 0.25% KCl (10:10:3, v/v).

                              
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Table III
Apparent Km and Vmax of pep-MPT with various peptide acceptor substrates

Substrate Specificity of pep-MPT-- The minimal requirements for pep-MPT activity were investigated using a number of peptide substrates. As shown in Table III, peptides with two Ser/Thr-rich repeats (i.e. sapp-1) were slightly better substrates than peptides with one Ser/Thr-rich repeat (i.e. sapp-4) (Km = 20 and 50 µM, respectively). Replacement of the four Ser residues in sapp-4 with Thr (sapp-5) virtually abrogated all substrate potential, confirming the specificity of MPT for Ser residues (Table III). Interestingly, replacement of the two acidic amino acids in the sequences flanking the serines (sapp-6) resulted in a 17-fold increase in the Km of the pep-MPT activity, suggesting that these residues may contribute to MPT substrate recognition (Table III). In the competition assay, unlabeled sapp-4 (1 mM) was slightly less effective than unlabeled sapp-1 (50% versus 55%) at reducing the rate of phosphoglycosylation of 3H-labeled sapp-1 (Fig. 6C). However, both sapp-5 and sapp-6 and a peptide containing 11 serine residues (Ser11 peptide) inhibited this reaction by <10%. These data suggest that replacement of Ser residues with Thr or removal of the acidic flanking residues in the repeat sequences abrogates recognition of the sAP-based peptides by pep-MPT.

Subcellular Distribution of pep-MPT-- Initial experiments showed that pep-MPT activity was sedimented in a 100,000 × g microsomal pellet and that activity was detected only when intact microsomal membranes were disrupted by sonication or detergent lysis, indicating that pep-MPT is a membrane-associated enzyme and that the catalytic domain has a luminal orientation. To investigate the subcellular location of this enzyme and thus the site at which protein phosphoglycosylation is initiated, L. mexicana microsomal membranes were prepared by nitrogen cavitation and fractionated by equilibrium velocity sucrose gradient centrifugation. Previously characterized markers for the endoplasmic reticulum (BiP) (29) and cis-Golgi apparatus (Rab1) (28) were separated on this sucrose gradient (Fig. 8), whereas analysis for other organelle-specific markers (i.e. hexose kinase (glycosomes), acid phosphatase (plasma membrane), and HSP60 (mitochondria)) showed that organelle separation had been achieved (data not shown). The pep-MPT activity displayed an overlapping (but not coincident) sedimentation pattern with Rab1 and was reproducibly separated by two fractions from the endoplasmic reticulum marker BiP. These data suggest that the phosphomannosylation of polypeptide acceptors is probably initiated in a post-endoplasmic reticulum compartment.


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Fig. 8.   pep-MPT colocalizes with cis-Golgi markers. L. mexicana promastigotes were lysed by nitrogen cavitation, and microsomes in a 3000 × g supernatant were resolved by sucrose gradient centrifugation. A, sedimentation profile of protein; B, sedimentation profile of the endoplasmic reticulum marker BiP, the cis-Golgi marker Rab1, and pep-MPT.

Differential Phosphoglycosylation of Exogenous Peptides and Endogenous Acceptors in Cell-free Systems-- Although the exogenous peptides are utilized by pep-MPT in in vitro assays, the addition of either unlabeled GDP-Man (data not shown) or UDP-Gal (Fig. 9, lane 1) to these assays did not result in further extension of the peptide-linked Man-PO4 residues with either alpha 1-2-linked mannose oligosaccharides or the phosphorylated Galbeta 1-4Man repeat units as occurs on endogenous sAP polypeptide acceptors (10). This was not due to disruption of the enzymatic machinery for phosphoglycan biosynthesis, as endogenous membrane-bound LPG acceptors were elaborated with at least one phosphodiester-linked disaccharide unit (Fig. 9, lane 3). This repeat unit probably corresponds to the backbone repeat unit PO4-6Galbeta 1-4Man, as the dephosphorylated species comigrated with authentic Galbeta 1-4Man on the HPTLC (Fig. 9, lane 3), and the synthesis of this oligosaccharide was dependent on the inclusion of UDP-Gal in the assay mixture (data not shown). Interestingly, the Triton X-100-insoluble pellet retained a significant amount of [3H]Man label (~30% of the non-peptide-associated activity), which could be released by mild acid treatment (Fig. 9, lane 2). As Triton X-100 quantitatively extracts both LPG and hydrophilic (non-lipid-linked) phosphoglycans,3 it is likely that most of the labeled material in the Triton X-100 pellet corresponds to tightly bound polypeptide acceptors. Unlike the endogenous LPG acceptors, the Triton X-100-insoluble acceptors were modified primarily with Man-PO4 and only to a small extent with the Galbeta 1-4Man disaccharide (Fig. 9, lane 2). Thus, this second pool of acceptors may have limited access to the enzymes involved in the synthesis of the disaccharide repeat units.


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Fig. 9.   Elongation of Man-1-PO4 on exogenous peptide and endogenous polypeptide and lipid acceptors in cell-free systems. Sonicated and washed L. mexicana membranes were suspended in buffer containing sapp-1 peptide, GDP-[3H]Man, and UDP-Gal. After incubation, membranes were sequentially extracted with water and Triton X-100 to remove exogenous peptides and Triton X-100-soluble LPG/phosphoglycans/glycoproteins, respectively. The 3H-labeled glycopeptides and LPG were further purified as described under "Experimental Procedures." This procedure generated three [3H]Man-labeled fractions containing exogenous peptide (lane 1), Triton X-100-insoluble glycoproteins (lane 2), and LPG (lane 3). Each of these fractions was subjected to mild acid hydrolysis (to selectively cleave phosphodiester-linked glycans) and then dephosphorylated with alkaline phosphatase, and the neutral glycans were analyzed by HPTLC.


    DISCUSSION

We have characterized a novel pep-MPT activity from L. mexicana promastigotes that is likely to be responsible for initiating the phosphoglycosylation of a major class of secreted glycoproteins and mucin-like molecules. The phosphoglycans may account for 20-90% of these secreted molecules and are structurally variable, ranging from single Man-PO4 residues to exceedingly complex and highly branched phosphoglycans, depending on the nature of the polypeptide backbone and parasite developmental stage (10, 14, 15). However, all these glycans are thought to be linked to the polypeptide backbone by a common Manalpha 1-PO4-Ser linker sequence (10, 14, 15). This linkage was indicated by the detection of O-phosphoserine in sAPs and PPGs and the resistance of these residues to alkaline phosphatase unless the masking glycans were removed with alpha -mannosidase or mild acid hydrolysis (10, 14, 15). However, it has been difficult to confirm the nature of this linkage by mass spectrometry or NMR, as the heavily glycosylated Ser/Thr-rich domains are essentially resistant to proteolysis, preventing the generation of small well defined glycopeptides that are amenable to such analyses. In this context, the synthesis of Manalpha 1-PO4-modified peptides by L. mexicana membranes and the characterization of these peptides by electrospray collision-induced dissociation mass spectrometry and chemical and enzymatic treatments have provided unequivocal confirmation of this novel linkage. Although the Manalpha 1-PO4-Ser linkage is apparently unique to leishmanial glycoconjugates, it is related to phosphodiester-linked glycans from other lower eukaryotic glycoconjugates. For example, the cysteine proteases of Dictyostelium discoideum are modified with GlcNAcalpha 1-PO4 residues linked to Ser, whereas secreted glycoproteins from Trypanosoma cruzi may be modified with structurally complex oligosaccharides containing a putative Xyl-1-PO4-Thr (Ser) linker sequence (11, 12). The abundance of this type of modification in several lower eukaryotes suggests that the glycan-PO4-Ser (Thr) motif may be more common than previously suspected.

We have provided evidence that L. mexicana pep-MPT transfers Manalpha 1-PO4 from GDP-Man to Ser-rich peptide sequences that are found in L. mexicana sAP. The transfer of Manalpha 1-PO4 from GDP-Man was supported by the finding that product formation was not inhibited by amphomycin, suggesting that dolichol-phosphate mannose is not utilized as a sugar donor by this enzyme. In addition, there was no evidence for the modification of the peptide in the absence of GDP-Man3 indicating that this linkage is not being assembled by the sequential action of a serine kinase followed by a novel mannosyltransferase. Based on ESI-MS product analysis and the use of various peptide acceptors, the following conclusions were made concerning the substrate specificity of pep-MPT. First, this enzyme only adds Manalpha 1-PO4 to Ser residues; a peptide containing Thr instead of Ser was neither a substrate nor an inhibitor of the enzyme. Second, the ESI-MS sequence analysis indicated that the Man-PO4 residues are added to several Ser residues within the Ser-rich repeats. Moreover, the partial characterization of peptides with up to five Man-PO4 residues shows that at least three of the four contiguous Ser residues in a single Ser-rich repeat sequence can be modified, consistent with the very high degree of substitution (~80%) observed on endogenous sAPs and PPGs (10, 14). Third, amino acids flanking the contiguous Ser sequences may be important for pep-MPT recognition and influence the pattern of substitution. For example, sAP peptides lacking the flanking Glu or Asp residues (i.e. sapp-6) were poorly utilized by pep-MPT in direct assays and were poor inhibitors in competition assays. It is possible that these residues may be required to maintain a suitable conformation or the solubility of the peptide, as polyserine peptides become increasingly insoluble above six residues in length.

The pep-MPT characterized here appears to be distinct from the two oligosaccharide-specific MPTs that are involved in adding Man-PO4 to the LPG anchor precursor (initiating MPT) or preformed phosphoglycan chains on lipid or peptide acceptors (elongating MPT) (20, 22). The initiating MPT can be assayed using either dephosphorylated LPG core or the structurally related oligosaccharide, stachyose, as acceptor (22). In contrast, the elongating MPT adds Manalpha 1-PO4 to the minimal disaccharide unit Galbeta 1-4Manalpha 1-PO4 that is present in the synthetic hydrophobic oligosaccharide acceptor L2 (20). Neither stachyose nor L2 inhibited the pep-MPT activity when present at 6-30-fold excess over the peptide acceptors (Fig. 6C). Moreover, unlabeled sapp-4 peptide did not inhibit the phosphomannosylation of L2 when present at concentrations that inhibit the phosphomannosylation of the 3H-labeled sapp-1 peptide (Fig. 7). pep-MPT could also be distinguished from the other MPTs by its solubility and stability in various detergents (20, 22). On the other hand, all three MPTs appear to utilize GDP-Man as the sugar donor and have a similar pH optimum and requirement for divalent cations. Although the substrate specificities of these MPTs appear to be distinct, it is notable that both pep-MPT and elongating MPTs are maximally active against substrates that contain negatively charged groups near the aglycon acceptor (i.e. acidic amino acids or phosphate, respectively) (this study and Ref. 20), suggesting that these activities may have arisen from a common progenitor enzyme.

Marked differences were observed in the levels of pep-MPT activity during promastigote growth, with 5- or 10-fold higher levels of activity being observed in rapidly dividing procyclic promastigotes compared with late stationary phase promastigotes and lesion-derived amastigotes, respectively. There is no evidence that the low pep-MPT activity of the metacyclic promastigotes or the intracellular amastigotes is associated with a reduced level of phosphoglycosylation of secreted polypeptides. Indeed, aPPGs are much more extensively modified with phosphoglycan chains than promastigote sAPs or promastigote PPGs (15). It is thus possible that the low pep-MPT activity in non-dividing promastigotes and the intracellular amastigotes may reflect a low level of secretory activity in these developmental stages or the presence of other pep-MPT activities that preferentially recognize distinct peptide sequences in aPPGs (14, 15).

Subcellular fractionation studies suggested that pep-MPT was localized in a post-endoplasmic reticulum compartment and that it had an overlapping (but not coincident) distribution with the Golgi marker Rab1. Rab1 has been localized primarily to cis-Golgi cisternae in L. major (28), but may also be localized to endoplasmic reticulum-Golgi transport vesicles, where it is thought to be involved in regulating the organization of vesicle proteins (38). The broader distribution of the Rab1 marker may reflect the partial distribution of this protein in lighter transport vesicles from which pep-MPT is excluded. Localization of pep-MPT to the Golgi apparatus is consistent with the recent demonstration that the leishmanial Golgi apparatus contains a GDP-Man transporter that is required for phosphoglycan biosynthesis (24). Similarly, the initiating and elongating MPTs involved in LPG biosynthesis are also thought to be localized to the Golgi apparatus based on the finding that one of the enzymes involved in LPG anchor biosynthesis has been localized to the Golgi apparatus by electron microscopy (23). In this respect, it is of interest that Man-PO4 residues on either the peptide or a pool of endogenous polypeptide acceptors were negligibly or poorly elaborated with phosphoglycan repeat units. In contrast, Man-PO4 residues on the endogenous LPG acceptors were quantitatively modified with at least one repeat unit. Although it is possible that the Man-PO4-modified peptide is a poor substrate for these elongating enzymes, the low rate of elongation of Man-PO4 on the endogenous polypeptide pool suggests that pep-MPT and elongating enzymes (including the elongating MPT) are localized to functionally distinct subcompartments of the Golgi apparatus. Compartmentalization of these enzymes may provide a mechanism for generating phosphoglycan structures with markedly different chain lengths on different classes of glycosylphosphatidylinositol or polypeptide acceptors (see Fig. 1).

In summary, we have confirmed the nature of the peptide linkage of the unusual phosphoglycans that are added to the major secretory proteins of L. mexicana and identified a novel pep-MPT activity. Similar types of phosphoglycosylation occur in all developmental stages of the parasite, including the amastigotes that perpetuate disease in the mammalian host and that have been shown to be functionally important. Consequently, it is likely that pep-MPT and possibly other MPTs are potential targets for new anti-leishmanial drugs.

    ACKNOWLEDGEMENTS

We thank Professor Michael Ferguson for providing the synthetic L2 acceptor, Drs. Emanuela Handman and James Bangs for antibodies, and Professor Tony Bacic for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by the Australian National Health and Medical Research Council and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Wellcome Trust Senior Research Fellow and to whom correspondence should be addressed. Tel.: 61-3-9344-5681; Fax: 61-3-9347-7730; E-mail: m.mcconville{at}biochemistry.unimelb.edu.au.

2 J. E. Ralton and M. J. McConville, unpublished data.

3 M. J. McConville, unpublished data.

    ABBREVIATIONS

The abbreviations used are: LPG, lipophosphoglycan; PPG, proteophosphoglycan; aPPG, amastigote proteophosphoglycan; sAP, secreted acid phosphatase; MPT, mannose-1-phosphotransferase; pep-MPT, peptide-specific mannose-1-phosphotransferase; CHAPS, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonic acid; RP-HPLC, reversed-phase high performance liquid chromatography; HPTLC, high performance thin-layer chromatography; ESI-MS, electrospray ionization mass spectrometry.

    REFERENCES
Top
Abstract
Introduction
References
  1. Turco, S. J., and Descoteaux, A. (1992) Annu. Rev. Biochem. 46, 65-94
  2. McConville, M. J., and Ferguson, M. A. J. (1993) Biochem. J. 294, 305-324[Medline] [Order article via Infotrieve]
  3. McConville, M. J., Schnur, L. F., Jaffe, C., and Schneider, P. (1995) Biochem. J. 310, 807-818[Medline] [Order article via Infotrieve]
  4. Ilg, T., Stierhof, Y.-D., Wiese, M., McConville, M. J., and Overath, P. (1994) Parasitology 108, S63-S71[Medline] [Order article via Infotrieve], and references therein
  5. Mengeling, B. J., Beverley, S. M., and Turco, S. J. (1997) Glycobiology 7, 873-880[Medline] [Order article via Infotrieve]
  6. Ilg, T., Stierhof, Y.-D., Etges, R., Adrian, M., Harbecke, D., and Overath, P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8774-8778[Abstract]
  7. Weise, M., Ilg, T., Lottspeich, F., and Overath, P. (1995) EMBO J. 14, 1067-1074[Abstract]
  8. Jaffe, C. L., Perez, L. M., and Schnur, L. F. (1990) Mol. Biochem. Parasitol. 41, 233-240[Medline] [Order article via Infotrieve]
  9. Ilg, T., Harbecke, D., Wiese, M., and Overath, P. (1993) Eur. J. Biochem. 217, 603-615[Abstract]
  10. Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081[Abstract/Free Full Text]
  11. Mehta, D. P., Ichikawa, M., Salimath, P. V., Etchison, J. R., Haak, R., Manzi, A., and Freeze, H. H. (1996) J. Biol. Chem. 271, 10897-10903[Abstract/Free Full Text]
  12. Haynes, P. A. (1998) Glycobiology 8, 1-5[Abstract/Free Full Text]
  13. Ilg, T., Stierhof, Y.-D., McConville, M. J., and Overath, P. (1995) Eur. J. Cell Biol. 66, 205-215[Medline] [Order article via Infotrieve]
  14. Ilg, T., Stierhof, Y.-D., Craik, D., Simpson, R., Handman, E., and Bacic, A. (1996) J. Biol. Chem. 271, 21583-21596[Abstract/Free Full Text]
  15. Ilg, T., Craik, D., Currie, G., Multhaup, G., and Bacic, A. (1998) J. Biol. Chem. 273, 13509-13523[Abstract/Free Full Text]
  16. Stierhof, Y.-D., Ilg, T., Russell, D. G., Hohenberg, H., and Overath, P. (1994) J. Cell Biol. 125, 321-331[Abstract]
  17. Peters, C., Kawakami, M., Ilg, T., Overath, P., and Aebischer, T. (1997) Eur. J. Immunol. 27, 2666-2672[Medline] [Order article via Infotrieve]
  18. Peters, C., Stierhof, Y.-D., and Ilg, T. (1997) Infect. Immun. 65, 783-786[Abstract]
  19. Carver, M. A., and Turco, S. J. (1991) J. Biol. Chem. 266, 10974-10981[Abstract/Free Full Text]
  20. Brown, G. M., Millar, A. R., Masterson, C., Brimacombe, J. S., Nikolaev, A. V., and Ferguson, M. A. J. (1996) Eur. J. Biochem. 242, 410-416[Abstract]
  21. Ng, K., Handman, E., and Bacic, A. (1996) Glycobiology 4, 845-853[Abstract]
  22. Mengeling, B. J., Zilberstein, D., and Turco, S. J. (1997) Glycobiology 7, 847-853[Abstract]
  23. Ha, D. S., Schwarz, J. K., Turco, S. J., and Beverley, S. M. (1996) Mol. Biochem. Parasitol. 77, 57-64[CrossRef][Medline] [Order article via Infotrieve]
  24. Ma, D., Russell, D. G., Beverley, S. M., and Turco, S. J. (1997) J. Biol. Chem. 272, 3799-3805[Abstract/Free Full Text]
  25. Bates, P. A., Hermes, I., and Dwyer, D. M. (1990) Mol. Biochem. Parasitol. 39, 247-256[CrossRef][Medline] [Order article via Infotrieve]
  26. Braell, W. A., Tyo, M. A., Krag, S. S., and Robbins, P. W. (1976) Anal. Biochem. 74, 484-487[Medline] [Order article via Infotrieve]
  27. Rush, J. S., Shelling, J. G., Zingg, N. S., Ray, P. H., and Waechter, C. J. (1993) J. Biol. Chem. 268, 13110-13117[Abstract/Free Full Text]
  28. Cappai, R., Osborn, A. H., Gleeson, P. A., and Handman, P. (1993) Mol. Biochem. Parasitol. 62, 73-82[CrossRef][Medline] [Order article via Infotrieve]
  29. Bangs, J. D., Uyetake, L., Brickman, M. J., Balber, A. E., and Boothroyd, J. C. (1993) J. Cell Sci. 105, 1101-1113[Abstract/Free Full Text]
  30. Tack, B. F., Dean, J., Eilat, D., Lorenz, P. E., and Schechter, A. N. (1980) J. Biol. Chem. 255, 8842-8847[Abstract/Free Full Text]
  31. Bates, P. A., Robertson, C. D., Tetley, L., and Coombs, G. H. (1993) Parasitology 105, 193-202
  32. Wessel, D., and Flugge, U. I. (1984) Anal. Biochem. 138, 141-143[Medline] [Order article via Infotrieve]
  33. Schneider, P., Ralton, J. E., McConville, M. J., and Ferguson, M. A. J. (1994) Anal. Biochem. 210, 106-112[CrossRef]
  34. Moritz, R. L., and Simpson, R. J. (1992) J. Chromatogr. 599, 119-130[CrossRef][Medline] [Order article via Infotrieve]
  35. Moritz, R. L., Eddes, J. S., Reid, G. E., and Simpson, R. J. (1996) Electrophoresis 17, 907-917[Medline] [Order article via Infotrieve]
  36. McConville, M. J., Thomas-Oates, J. E., Ferguson, M. A. J., and Homans, S. W. (1990) J. Biol. Chem. 265, 19611-19623[Abstract/Free Full Text]
  37. Annan, R. S., and Carr, S. A (1996) Anal. Chem. 68, 3413-3421[CrossRef][Medline] [Order article via Infotrieve]
  38. Pind, S. N., Nuoffer, C., McCaffery, J. M., Plutner, H., Davidson, H. W., Farquhar, M. G., and Balch, W. E (1994) J. Cell Biol. 125, 239-252[Abstract]


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