From the Plant Cell Biology Research Centre, School
of Botany, University of Melbourne, Victoria 3052, Australia, the
§ Walter and Eliza Hall Institute of Medical Research,
P. O. Royal Melbourne Hospital, Victoria 3050, Australia, the
Centre for Drug Design and Development, University of
Queensland, Brisbane, Queensland 4072, Australia, and the ** Centre for
Molecular Biology, Heidelberg, Federal Republic of Germany
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ABSTRACT |
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Intracellular amastigotes of the
protozoan parasite Leishmania mexicana secrete a
macromolecular proteophosphoglycan (aPPG) into the phagolysosome of
their host cell, the mammalian macrophage. The structures of aPPG
glycans were analyzed by a combination of high pH anion exchange high
pressure liquid chromatography, gas chromatography-mass spectrometry,
enzymatic digestions, electrospray-mass spectrometry as well as
1H and 31P NMR spectroscopy. Some glycans are
identical to oligosaccharides known from Leishmania
mexicana promastigote lipophosphoglycan and secreted acid
phosphatase. However, the majority of the aPPG glycans represent
amastigote stage-specific and novel structures. These include neutral
glycans ([Glc1-3]1-2Gal
1-4Man,
Gal
1-3Gal
1-4Man, Gal
1-3Glc
1-3Gal
1-4Man), several
monophosphorylated glycans containing the conserved phosphodisaccharide
backbone (R-3-[PO4-6-Gal]
1-4Man) but carrying
stage-specific modifications (R = Gal
1-,
[Glc
1-3]1-2Glc
1-), and monophosphorylated aPPG
tri- and tetrasaccharides that are uniquely phosphorylated on the
terminal hexose (PO4-6-Glc
1-3Gal
1-4Man, PO4-6-Glc
1-3Glc
1-3Gal
1-4Man,
PO4-6-Gal
1-3Glc
1-3Gal
1-4Man). In addition aPPG
contains highly unusual di- and triphosphorylated glycans whose major
species are
PO4-6-Glc
1-3Glc
1-3[PO4-6-Gal]
1-4Man, PO4-6-Gal
1-3Glc
1-3[PO4-6-Gal]
1-4Man,
PO4-6-Gal
1-3Glc
1-3Glc
1-3[PO4-6-Gal]
1-4Man, PO4-6-Glc
1-3[PO4-6-Glc]
1-3[PO4-6-Gal]
1-4Man,
PO4-6-Gal
1-3[PO4-6-Glc]
1-3Glc
1-3[PO4-6-Gal]
1-4Man, and
PO4-6-Glc
1-3[PO4-6-Glc]
1-3Glc
1-3[PO4-6-Gal]
1-4Man.
These glycans are linked together by the conserved phosphodiester
R-Man
1-PO4-6-Gal-R or the novel
phosphodiester R-Man
1-PO4-6-Glc-R and are connected to
Ser(P) of the protein backbone most likely via the linkage R-Man
1-PO4-Ser. The variety of stage-specific glycan
structures in Leishmania mexicana aPPG suggests the
presence of developmentally regulated amastigote
glycosyltransferases which may be potential anti-parasite drug
targets.
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INTRODUCTION |
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Protozoan parasites of the genus Leishmania are the causative agent of a spectrum of human diseases. Leishmania have a digenetic life cycle that encompasses the extracellular promastigotes in the digestive tract of the parasite-transmitting insect vector, the sandfly, and the disease-causing intracellular amastigotes living in parasitophorous vacuoles of mammalian macrophages.
The parasites produce unusual glycoconjugates, which are thought to
play crucial roles for survival, development, and virulence in both
developmental stages of the parasite. The best characterized Leishmania glycoconjugate is the promastigote cell-surface
glycolipid, lipophosphoglycan
(LPG).1 The structure of LPG
from five different Leishmania species has been determined.
LPG contains a conserved lyso-alkylphosphatidylinositol membrane anchor linked to a phosphohexasaccharide core structure, a
conserved backbone of up to 40 phosphodiester-linked disaccharide repeats (PO4-6-Gal1-4Man
1-), and species-, strain-,
and stage-specific components linked to the core and repeats as well as
terminating neutral (cap) glycans at the non-reducing end of the
molecule (reviewed in Refs. 1 and 2). In the sandfly, LPG serves as a
ligand for the attachment of non-infectious procyclic promastigotes to
the midgut wall lining and may protect the parasites against the
hydrolytic environment of the insect's digestive tract. A stage-specific form of LPG confers complement resistance to the highly
infectious metacyclic promastigotes, which are injected by the sandfly
into the skin of the mammalian host. LPG also acts as a receptor for
the invasion of macrophages by metacyclic promastigotes and may protect
this transient mammalian parasite stage against the initial
microbicidal response of the host cell by acting as a radical scavenger
and by modulating signal transduction and gene expression of the
macrophage (reviewed in Refs. 3-5). Structure-function analysis of LPG
and its fragments demonstrated the importance of the glycolipid anchor,
the phosphoglycan chains, and the cap oligosaccharides for these
functions (6-13). The biosynthetic pathway of LPG has been partially
elucidated and has been implicated as a potential target for the
development of chemotherapeutic agents (14-20).
Although the crucial role of LPG for the promastigote stages of Leishmania is well established, its importance for the disease-causing amastigote stage in the mammalian host is less clear. Leishmania donovani and Leishmania mexicana amastigotes do not express LPG (21, 22). Leishmania major amastigotes synthesize low levels of a stage-specific LPG (23-25), which may be involved in host cell binding and uptake (26), but amastigote LPG does not form a protective surface glycocalyx like in promastigotes (27).
It has been demonstrated that some of the biologically active structural elements of LPG like the repetitive phosphoglycans and the neutral cap oligosaccharides are also present on Leishmania promastigote proteins like acid phosphatase (sAP) (28-33) and the filamentous proteophosphoglycan of promastigotes (pPPG) (32, 34). In these molecules, the glycans are linked to the protein backbone via phosphoserine residues (33, 34), a form of protein glycosylation not yet observed in mammalian cells. Recently, it has been shown that protein-bound phosphoglycans are also present in the amastigote stage of L. mexicana (22, 35). This amastigote proteophosphoglycan (aPPG) is secreted by the parasites in large amounts into the phagolysosomes of host macrophages, where it may accumulate to mg/ml concentrations (35). The massive secretion of aPPG by amastigotes may contribute to the expansion of the phagolysosomes to huge parasitophorous vacuoles, which are the hallmark of L. mexicana infections (36). It has also been demonstrated that L. mexicana aPPG is an activator of the complement cascade via the lectin pathway. This property may contribute to lesion development and pathology caused by L. mexicana (37). A preliminary analysis of aPPG showed that it is immunologically and chemically related to the promastigote phosphoglycan antigens LPG and sAP but also exhibits distinct properties (35).
In the present study we describe the structural analysis of the glycans from L. mexicana aPPG purified from infected mouse lesion tissue. We demonstrate that, in addition to oligosaccharides also present in L. mexicana promastigote phosphoglycan antigens, aPPG contains a variety of stage-specific neutral and phosphorylated glycans. These glycans are linked together by phosphodiester bonds and are most likely connected to the protein backbone via phosphoserine residues. We also show that a large proportion of aPPG glycans is modified by two, three, or even four phosphate groups in diester linkages to other glycans.
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EXPERIMENTAL PROCEDURES |
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Parasites-- L. mexicana promastigotes (strain MNYC/BZ/62/M379) were grown in semi-defined medium 79 as described (38). L. mexicana amastigote-infected tissue was obtained from dorsal lesions of CBA mice infected 3-6 months previously with 5 × 106 stationary phase promastigotes in the shaven rump at the base of the tail.
Purification of Phosphoglycan Antigens and Analytical Procedures-- L. mexicana aPPG was purified from infected mouse tissue as described earlier (35) with the modifications reported recently (36). Purification of L. mexicana LPG, sAP, PG, pPPG, and L. major pPPG from promastigote culture supernatant was performed as described previously (33, 34, 39).
Analytical Techniques--
Colorimetric carbohydrate, protein,
and phosphate analysis, mild acid hydrolysis, 40% HF
dephosphorylation, phosphoamino acid analysis, and SDS-polyacrylamide
gel electrophoresis were performed as outlined previously (31, 33, 34,
39). Carbohydrate in Superose 6 fractions was detected
semi-quantitatively by spotting 1-µl aliquots onto Silica Gel 60 plates (Merck, Darmstadt, FRG) followed by reaction with
orcinol/H2SO4 (39). Two-site ELISA using the
monoclonal antibody AP3 as trapping antibody was also performed as
described before (35), except for using biotinylated AP3 followed by
Extravidin coupled to calf intestine alkaline phosphatase (AP) (Sigma,
Deisenhofen, FRG) or LT22 followed by goat anti-mouse IgG
(-chain-specific) antibodies coupled to AP (Sigma) as detection
systems. Combined gas chromatography-mass spectometry (GC-MS) was
performed using a Hewlett-Packard HP6859 GC-MS, fitted with either a
25-m × 0.3-mm CPSil5 low polarity column (Chrompack, Middleburg,
The Netherlands) for trimethylsilyl derivatives and permethylated
alditol acetates or a 25-m × 0.22-mm BPX-70 high polarity column
(SGE, Ringwood, Australia) for alditol acetates and permethylated
alditol acetates as described previously (34). Amino acid analysis and
N-terminal protein sequencing were performed on automated systems
(Applied Biosystems, models 420A and 477A, respectively) according to
the manufacturer's protocols.
Enzyme Treatment of Glycans--
Neutral glycans were treated
with sweet almond -glucosidase (SABG, Boehringer Mannheim, FRG, 40 units/ml), jack bean
-mannosidase (JBAM, Sigma, 10 units/ml), or
bovine testes
-galactosidase (BTBG, Boehringer Mannheim, 0.2 units/ml) in 10 mM sodium acetate, pH 4.5, for 16 h at
37 °C. Dephosphorylation of phosphoglycans by AP (50 units/ml) was
performed in 20 mM NH4HCO3 for
4-16 h at 37 °C. Samples were either desalted (34) or diluted 1:20
and then rechromatographed by high pH anion exchange HPLC
(HPAE-HPLC).
Monosaccharide and Methylation Analysis-- Native and mild acid-hydrolyzed aPPG (10 µg) containing myo-inositol as an internal standard and monosaccharide standards were subjected to methanolysis, re-N-acetylated, and following trimethylsilylation analyzed by GC-MS either directly or after methylation with diazomethane (40) as described previously (25, 39). Alternatively, neutral monosaccharides of aPPG and of purified oligosaccharides (0.5-10 µg) were also determined as alditol acetates prepared and analyzed by standard methods (41). To quantitate neutral and phosphorylated monosaccharides in L. mexicana sAP and L. mexicana aPPG, samples (10 µg) were hydrolyzed in 2 M trifluoroacetic acid for 2 h at 105 °C. This treatment cleaves most glycosidic bonds involving pentoses and hexoses quantitatively (41), whereas the phosphomonoester bonds of hexose 6-phosphates are stable. The resulting neutral and phosphorylated monosaccharides were separated by HPAE-HPLC using program 8 (see below). Pooled fractions were desalted by passage over AG50 × 12 (H+) and lyophilized; myo-inositol was added as an internal standard, and the phosphorylated glycans were dephosphorylated by AP treatment (see above). The resulting monosaccharides were reduced, acetylated, and analyzed as alditol acetates by GC-MS and quantitated relative to the internal standard. Methylation linkage analysis of dephosphorylated glycans (0.5-5 µg) was performed as described previously (25, 39, 43).
High pH Anion Exchange HPLC-- Neutral and phosphorylated glycans released from L. mexicana aPPG by mild acid hydrolysis or 40% HF treatment were separated by high pH anion exchange HPLC on a Dionex BioLC carbohydrate analyzer (Dionex Corp. Sunnyvale, CA) using a Carbo-Pac PA1 column and pulsed amperometric detection using linear gradients of sodium acetate in 150 mM NaOH. Several gradient programs were used as follows: program 1, 0 mM for 6 min, raised to 50 mM over 18 min, to 125 mM over 7 min, and held at 125 mM for 14 min; program 2, program 1 followed a raise of the sodium acetate concentration to 175 mM over 3 min, to 250 mM over 30 min, held at 250 mM for 10 min, raised to 625 mM over 10 min, and held at 625 mM for 5 min; program 3, 0 mM for 6 min, raised to 50 mM over 18 min, to 125 mM over 21 min, and held at 125 mM for 15 min; program 4, 250 mM for 6 min, raised to 1000 mM over 1 min, and held at 1000 mM for 13 min; program 5, 250 mM for 6 min, raised to 385 mM over 1 min, held at 385 mM for 18 min, raised to 625 mM over 1 min, and held at 625 mM for 10 min; program 6, isocratic, 385 mM for 20 min; program 7, 385 mM for 15 min, raised to 625 mM over 30 min, and held at 625 mM for 10 min. For the separation of hexoses from hexose phosphates, the Carbo-Pac PA1 column was held at 100 mM NaOH for 10 min; NaOH and NaAc were then raised to 150 and 187.5 mM, respectively, over 2 min and held at that concentration for 18 min (program 8).
Electrospray Ionization-Mass Spectrometry (ES-MS)-- Mass spectra of oligosaccharides were acquired on a Finnigan LCQ ES-MS. Samples were introduced into the electrospray source through a rheodyne injector with a 5-µl loop at a flow rate of 5 µl/min in either 25% methanol in H2O for native oligosaccharides or 50% aqueous acetonitrile for permethylated oligosaccharides. Mass spectra were acquired both in the negative and the positive ion mode using the following conditions. The heated capillary was set to 170 °C; the maximum trapping time was 500 ms; the capillary, tube lens, and needle voltages were 25, 50, and 4.5 V, respectively, and the number of microscans was set to 1. MS-MS scans were performed by trapping the ion of interest and ejecting all other ions outside a 3-atomic mass unit window centered around the parent ion. Collision energy was set such that the parent ion was attenuated between 95 and 99%. Data were collected as averages of four spectra.
NMR Spectroscopy-- NMR spectra were recorded in D2O on Bruker ARX 500 or DMX 750 spectrometers for the intact aPPG, LPG, and a range of neutral and phosphorylated fragment glycans. Most spectra were recorded at 288 K, but selected spectra were also recorded at temperatures in the range 296 to 310 K to check for the presence of anomeric signals coincident with the residual solvent resonance. Spectra included one-dimensional 1H and 31P NMR, two-dimensional 1H-1H TOCSY, double quantum-filtered correlation spectroscopy, nuclear Overhauser effect spectroscopy, rotating-frame nuclear Overhauser effect spectroscopy spectra, and 31P-1H HMBC spectra. Some 1H spectra of phosphorylated glycans were recorded in the presence and absence of 31P decoupling. The one- and two-dimensional 31P spectra were recorded at 202 MHz, whereas all 1H NMR spectra were recorded at either 500.13 or 750.13 MHz. One-dimensional 1H spectra at 500 MHz were recorded with a spectral width of 5050 Hz, a pulse length of 7.5 µs (60°), and a relaxation delay between scans of 3 s. Similar conditions were used for the spectra recorded at 750 MHz. Typically 256-512 scans, for weaker samples up to several thousand scans, were accumulated. Mild presaturation was used to suppress the residual HOD signal from the D2O solvent. Spectra were processed using an exponential line broadening function of 0.3 Hz. Chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate at 0.00 ppm. One-dimensional 31P spectra were recorded with a spectral width of 5000 Hz, a pulse width of 5 µs (45°), and a relaxation delay between scans of 2 s. Typically 500 scans were acquired prior to Fourier transformation. Spectra were processed using an exponential line broadening function of 5-10 Hz. Chemical shifts were referenced to an external capillary of neat H3PO4 at 0.00 ppm. All two-dimensional homonuclear 1H spectra were recorded at 750 MHz, typically with spectral widths of 2500 Hz in both dimensions, 4K data points in frequency dimension F2 and 256-800 increments in F1, each of 16-256 scans. TOCSY spectra were recorded with a mixing time of 80 ms, while for nuclear Overhauser effect spectroscopy and rotating-frame nuclear Overhauser effect spectroscopy of selected glycans the mixing times were 300 and 250 ms, respectively. In most cases gradient pulses using the Watergate sequence (42) were used to suppress the residual solvent resonance, but in selected cases presaturation was used. The two-dimensional data were processed as a 4094 × 2048 data matrix, generally using shifted sine-bell apodization functions in both dimensions. HMBC spectra were recorded using 2048 complex data points in F2. A total of 32 scans was recorded for each of 128 slices over a spectral width of 800 Hz in F1. A relaxation delay of 1.0 s was used between scans, with an evolution delay of 50 ms in the HMBC sequence, as described previously (34). The two-dimensional data were processed as a 2048 × 1024 matrix, with a squared sine bell window function applied in both dimensions.
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RESULTS |
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L. mexicana aPPG Glycans Are Linked to Phosphoserine of the Protein Backbone via Mild Acid-labile Phosphodiester Bonds-- L. mexicana aPPG purified from amastigote-infected mouse lesion tissue eluted on a Superose 6 gel filtration column as a broad peak between the 2000 and the 440 kDa markers as detected by phosphate and carbohydrate determination as well as two-site ELISA (Fig. 1A). On SDS-polyacrylamide gels aPPG was detected in positive fractions as a smear migrating above the 200-kDa marker protein (Fig. 2A, lane 1 and Fig. 2B, lanes 9-14). After mild acid hydrolysis, known to be selective for hexose 1-phosphate bonds (43, 44), the high molecular weight aPPG (Fig. 2A, lane 1) disappeared on SDS-polyacrylamide gels, and a series of polypeptide bands between 40 kDa and the gel front were detected (Fig. 2A, lane 2). In Superose 6 chromatography, the majority of the carbohydrate (>95%) and phosphate (>90%) of mild acid-treated aPPG was found near the inclusion volume (Vt) of the column with small amounts of phosphate and traces of glycans in a second earlier eluting peak (Fig. 1B, fractions 15-18). This peak contained the polypeptides released by mild acid (Fig. 2C, lanes 15-19), which were pooled and subjected to amino acid analysis and protein sequencing. The amino acid composition of the pooled polypeptides (2.8% Asp/Asn, 5.7% Glu/Gln, 28.9% Ser, 11% Gly, 3.3% His, 1.2% Arg, 9.9% Thr, 9.7% Ala, 7.7% Pro, 1.6% Tyr, 4.9% Val, 2.1% Ile, 3.7% Leu, 6.9% Phe, and 0.6% Lys) was similar to the published composition of intact aPPG (35), which suggests that they correspond to the deglycosylated aPPG protein backbone. N-terminal sequencing of the pooled polypeptides and one of the Superose 6 fractions (Fig. 2C, fraction 16) showed in both analyses the peptide sequence NPIFXXD (where X indicates ambiguities). This indicates that despite the complex pattern on SDS-polyacrylamide gels (Fig. 2A, lanes 2 and 4 and Fig. 2C, lanes 15-19), the aPPG protein backbone may be formed by either one or several closely related polypeptide species. Phosphoamino acid analysis showed that only serine residues are phosphorylated (>25%). Phosphoserine in aPPG was resistant to mild acid deglycosylation and to AP treatment, whereas the consecutive application of both treatments led to the loss (>90%) of the phosphorylated amino acid. On SDS-polyacrylamide gels, the polypeptides obtained after mild acid deglycosylation of aPPG were readily visualized by the cationic dye Stains-all (Fig. 2A, lane 2) with an intense blue color, and after dephosphorylation no staining occurred (Fig. 2A, lane 3). Coomassie Blue staining of the same gel revealed the dephosphorylated polypeptides between 65 and 50 kDa apparent molecular mass (Fig. 2A, lane 5). Taken together the results indicated that the majority of aPPG glycans are linked to a serine-rich protein backbone via mild acid labile phosphodiester bonds to serine. This interpretation was corroborated by 31P NMR spectroscopy, which shows that phosphate is exclusively present in diester linkages in intact aPPG (see below).
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The Majority of the Mild Acid Labile L. mexicana aPPG Glycans Are Stage-specific-- The mild acid-released aPPG glycans (Fig. 1B, fractions 20-24) were separated by HPAE-HPLC under conditions which resolve neutral, monophosphorylated, and multiply phosphorylated glycans (Fig. 3C). Mild acid-released glycans of L. mexicana promastigote LPG (Fig. 3A) and sAP (Fig. 3B) served as standards and were resolved under identical conditions. Whereas L. mexicana aPPG contains the entire set of cap glycans and monophosphorylated glycans previously identified in LPG and sAP, the majority of its oligosaccharides are amastigote stage-specific and not detected in the promastigote phosphoglycan antigens.
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Structural Analysis of the Neutral Cap Oligosaccharides and the
HF-dephosphorylated Oligosaccharide Backbones of L. mexicana
aPPG--
The mild acid-released neutral aPPG cap oligosaccharides and
the neutral glycans obtained by 40% HF dephosphorylation of intact aPPG (corresponding to the entire set of oligosaccharide backbones) were isolated for structural analysis by HPAE-HPLC (compare Fig. 3C (0-48 min) and Fig.
4A, respectively). Their
structures were determined by monosaccharide analysis, methylation
linkage analysis, ES-MS (Table I),
exoglycosidase digests (Table I, compare also Fig. 4, B and
C), and coelution with authentic standards (Fig. 3,
A and B). The results are summarized in Table
II;
Man,2 the
manno-oligosaccharide series N2a-N6a ((Man1-2)1-5Man), N2b (Gal
1-4Man), and N3c (Glc
1-3Gal
1-4Man) are known
components of L. mexicana promastigote LPG and sAP (33, 39).
N3b (Gal
1-3Gal
1-4Man) and N4c
(Glc
1-3Glc
1-3Gal
1-4Man) were previously identified in
L. major and Leishmania tropica LPG (2, 43) but
not in L. mexicana glycoconjugates. N4b, N5b-d, N6b, and
N6c, however, represent completely novel glycan backbones. BTBG
digestion (product N3c), SABG
digestion3 (product N2b),
ES-MS of permethylated samples, and methylation analysis of N4b
resulted in the proposed structure Gal
1-3Glc
1-3Gal
1-4Man (Tables I and II, Fig. 4). The same analysis of N5b and N5d suggested the structures Gal
1-3Glc
1-3Glc
1-3Gal
1-4Man and
Glc
1-3Glc
1-3Glc
1-3Gal
1-4Man, respectively. N5c may be
either Glc
1-3Gal
1-3Glc
1-3Gal
1-4Man or
Glc
1-3Glc
1-3Gal
1-3Gal
1-4Man, which cannot be
distinguished by the methods used. A similar situation arises with N6b
and N6c, which have most likely the structure
Glc
1-3Hex
1-3Hex
1-3Hex
1-3Gal
1-4Man, where two of the
hexoses are Glc and one is Gal (Tables I and II, Fig. 4). The dominant
glycan backbone in aPPG is N4c followed by Man, N2b, N4b, and N3c. In
contrast in the two promastigote phosphoglycan antigens L. mexicana LPG and sAP, N2b and N3c are the major glycans (70-90
mol %; Table II and Ref. 33). Stage-specific backbone structures (N3b,
N4b,c, N5b-d, N6b and -c) not previously observed in either of the
promastigote glycoconjugates form the majority of the aPPG glycans
(>55 mol %).
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L. mexicana aPPG Contains Gal-6-PO4 and Glc-6-PO4-- Phosphohexose analysis of L. mexicana aPPG was performed using L. mexicana sAP as a reference compound. Qualitative analysis (GC-MS) showed that aPPG contains both Gal-6-PO4 and Glc-6-PO4, whereas sAP contains only Gal-6-PO4. None of the samples contained Man-6-PO4. To quantitate the degree of hexose phosphorylation in the two compounds, aPPG and sAP were hydrolyzed in 2 M trifluoroacetic acid; neutral and phosphorylated monosaccharides were separated by HPAE-HPLC and their compositions were analyzed after AP digestion, reduction, and acetylation as alditol acetates by GC-MS (Fig. 5). L. mexicana sAP contained only Gal-PO44 (Fig. 5A) and exhibited a Hex:Hex-PO4 ratio of 4.2:1. In contrast, L. mexicana aPPG contained both Gal-PO44 and Glc-PO44 (Fig. 5B) at a ratio of 1.4:1, and its Hex:Hex-PO4 ratio was 2.6:1.
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L. mexicana aPPG Contains Conserved and Novel Monophosphorylated
Glycans and Novel Di- and Triphosphorylated
Oligosaccharides--
HPAE-HPLC of mild acid-released aPPG glycans
resulted in a variety of peaks in the region of the salt gradient where
monophosphorylated glycans are expected to elute (Mono-P region, Fig.
3C). A further increase in salt concentration eluted two
unresolved glycan peak areas (Di-P and
Tri-/Tetra-P region, Fig. 3C) that are not
observed in L. mexicana LPG and sAP (compare Fig. 3,
A and B). All the aPPG phosphorylated glycans
(Fig. 3C) were sensitive to AP, and the corresponding
dephosphorylation products coeluted with the neutral glycans N2b,
N3b-c, N4b-c, N5b-d, and N6b-c (Fig.
6A). The structures of these
glycan backbones were confirmed by ES-MS of permethylated samples and
methylation linkage analysis (Table II). The much higher complexity and
the presence of novel structures in L. mexicana aPPG
versus LPG were also apparent in the comparison of the ES-MS
(M H)
pseudomolecular ions of mild acid
hydrolysates. Whereas LPG gave rise only to the expected ions for
Hex2P and Hex3P (not shown), aPPG showed, in
addition, ions for phosphorylated tetra- and pentasaccharides (Hex4P and Hex5P, Fig.
7A). Surprisingly the most
abundant molecular species in ES-MS of aPPG corresponded to novel
diphosphorylated tetrasaccharides (Hex4P2, Fig.
7A). In addition diphosphorylated pentasaccharides
(Hex5P2) and triphosphorylated pentasaccharides (Hex5P3) were detected (Fig.
7A).
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Monophosphorylated aPPG Glycans: Structure of the Major Components
and Identification of Novel Alternative Phosphorylation Sites--
The
major glycans and most of the minor glycans from the relatively well
resolved Mono-P glycan region in HPAE-HPLC (Fig. 3C) were
isolated and their structure determined by a combination of negative
ion ES-MS and ES-MS-MS (Table III, Fig.
8) and coelution on HPAE-HPLC with
standard compounds before and after AP treatment (not shown). These
analyses suggest that the structures of P2 and P3c are
PO4-6-Gal1-4Man and
Glc
1-3(PO4-6-Gal)
1-4Man, respectively, identical to
the respective glycans of L. mexicana LPG (39) and sAP (33).
NMR spectroscopy confirmed the structure of P2 (not shown) and P3c. The
downfield signals (at 5.18, 4.89, 4.68, 4.51, and 4.25 ppm) of the
one-dimensional 1H NMR spectrum of P3c are schematically
marked on Fig. 9A and are
discussed as a basis for the interpretation of structurally related but
novel aPPG glycans (see below); the two signals at 5.18 and 4.89 ppm
are assigned to H-1 of the reducing Man residue. The signal at 4.51 ppm
is due to H-1 of the phosphorylated Gal residue. The TOCSY spectrum
shows that this signal is part of the same spin system as the peak at
4.25 ppm which corresponds to the H-4 shift of the phosphorylated Gal
by analogy with P3 from L. major LPG (43). The small
coupling observed on the peak at 4.25 ppm is consistent with a Gal, as
H-4 has only gauche couplings with H-3 and H-5 (in contrast to Glc
where H-4 is axial). A lack of heteronuclear splitting on this peak and
its downfield shift (~0.06 ppm from H-4 in neutral glycans (43)) is
also consistent with phosphorylation at C-6. The remaining anomeric
signal at 4.68 ppm with a large coupling is assigned to the terminal
-Glc residue (33, 43).
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Structure of Novel Diphosphorylated aPPG Glycans--
ES-MS of the
unresolved aPPG glycans eluting in the first large peak under high salt
conditions in HPAE-HPLC (Di-P, Fig. 3C) revealed
two major ion species corresponding to the novel diphosphorylated glycans Hex4P2 and
Hex5P2 (not shown, see also Fig.
7A). After dephosphorylation the neutral glycan backbones
were purified by HPAE-HPLC (Fig. 6B) and analyzed by
positive ion ES-MS of the permethylated glycans as well as by
methylation linkage analysis. The major oligosaccharide corresponded to
N4c, whereas minor glycan backbones were N4b, N5b, and N5d (Table II).
Individual diphosphorylated glycans were separated by HPAE-HPLC under
isocratic conditions. Three glycans were obtained in a purified form
(>90%), Di-P4b, Di-P4c, and Di-P5b (Fig. 3D), whose
dephosphorylation products corresponded to N4b, N4c, and N5b,
respectively (Table II). The main fragmentation series in ES-MS-MS
(Fig. 7C, Fig. 8, and Table III) together with the absence
of Man-PO4 in the hexose phosphate analysis (Fig.
5B) suggested the structures
PO4-Gal1-3Glc
1-3(PO4-Gal)
1-4Man for
Di-P4b and
PO4-Glc
1-3Glc
1-3(PO4-Gal)
1-4Man for
Di-P4c (Table IV). These results were confirmed and extended by
1H NMR, 31P NMR, and HMBC spectroscopy on
Di-P4b' and Di-P4c (Fig. 9, C-F). Phosphorylations at C-6 of both the Gal residue and the terminal Glc in
Di-P4c are indicated by the characteristic downfield shift of Gal H-4
(4.25 ppm) and the two multiplet signals for the 6-CH2 protons of the terminal Glc (Fig. 9C) at 4.05 and 4.18 ppm.
These two multiplets simplify on 31P decoupling, confirming
a Glc-6-CH2-O-P linkage. The Di-P4b glycan is different
from Di-P4c in that the terminal Glc is replaced by Gal. As expected,
the characteristic Glc-6-CH2-O-P proton signals are missing
from the Di-P4b spectrum (Fig. 9D). The HMBC spectra of
these two diphosphorylated derivatives (Fig. 9, E and
F), which show cross-peaks only for those protons which have
a long range spin-spin coupling interaction with 31P nuclei
(in this case three bond couplings, compare Ref. 34), confirm these
proposed assignments. For Di-P4b two signals are detected, with
31P shifts characteristic of monoester phosphate groups and
1H shifts characteristic of Gal 6-CH2. These
signals occur at 4.02 and 3.98 ppm for the two Gal residues, each
corresponding to two near-degenerate CH2 chemical shifts
(Fig. 9F). By contrast, in Di-P4c the individual
Glc-6-CH2 protons have different chemical shifts, and this
leads to two signals (each split by H-H coupling) as indicated in Fig.
9E. The Gal-6-CH2 protons remain close to degenerate and yield a single cross-peak. The fact that a single 31P nucleus yields HMBC cross-peaks to two protons
unequivocally confirms the phosphorylation site for the terminal Glc to
be at 6-CH2 rather than at one of the ring positions,
which have only single protons.
Structure of Triphosphorylated Glycans and Evidence for
Tetraphosphorylated Glycans--
The aPPG glycans of the second
HPAE-HPLC high salt peak (Tri- and Tetra-P, Fig.
3C) were rechromatographed to remove residual diphosphorylated glycans and to resolve the triphosphorylated glycans
(Tri-P, Fig. 3E) from other components. ES-MS on
the pooled triphosphorylated glycans revealed three major ions
corresponding to Hex4P3 (905.8 atomic mass
units), Hex5P3 (1068.5 atomic mass units), and
Hex6P3 (1229.7 atomic mass units) (not shown,
compare also Fig. 7A). After dephosphorylation, N4c, N5b,
N5c, N5d, N6b, and N6c (Table II) were detected in HPAE-HPLC (Fig.
6C), and their structures were confirmed by methylation
analysis. The ES-MS-MS fragmentation pattern of Tri-P4c from the native
glycan mixture (Table III and Fig. 8) was consistent with the structure
PO4-Glc1-3(PO4-Glc)
1-3(PO4-Gal)
1-4Man (Table IV). Partial purification of the pooled glycans yielded three
fractions (fractions 1-3, Fig. 3E). Fraction 3 contained mainly Tri-P5d and only small amounts of Tri-P6b as shown by AP digestion followed by HPAE-HPLC (not shown). ES-MS-MS on Tri-P5d (Table
III and Fig. 8), the results of the hexose phosphate analysis and
methylation analysis indicated the structure
PO4-Glc
1-3(PO4-Glc)
1-3Glc
1-3(PO4-Gal)
1-4Man. ES-MS-MS spectra of the Hex5P3
molecular ion of the mixture of triphosphorylated glycans or fractions
enriched for either Tri-P5b or TriP5c (relative abundance, fraction 2:
Tri-P5b = TriP5c; fraction 1: Tri-P5b > Tri-P5c) were very
similar to that of Tri-P5d (Table III and Fig. 8), suggesting that the
structures of Tri-P5b and Tri-P5c could be
PO4-Gal
1-3(PO4-Glc)
1-3Glc
1-3(PO4-Gal)
1-4Man and
PO4-Glc
1-3(PO4-Hex)
1-3Hex
1-3(PO4-Gal)
1-4Man,
respectively. The fragment ions for Hex3P3 (725 and 743 atomic mass units) that were present in all Tri-P5 spectra at
low abundance (Table III) were most likely PO4 migration
products as discussed above for the Di-P glycans.5,7
1H and 31P NMR Spectroscopy on Intact
aPPG--
The one-dimensional 1H and 31P NMR
spectra, 31P-1H HMBC spectra of L. mexicana aPPG in D2O, and for comparison some
corresponding spectra of L. mexicana LPG are shown in Fig.
10. The assignments in the displayed
region of the 1H NMR spectrum of aPPG (Fig. 10A)
are based on the accumulated information from the component glycans,
the HMBC spectra, and relevant literature data with one exception. In
the one-dimensional 1H NMR spectrum the peak at 5.30 ppm is
similar to one previously assigned to Man1-PO4 in sAP
based on comparison with earlier shifts (33). However it appears likely
that this assignment was incorrect. Acquisition of the spectrum of aPPG
in the presence of 31P decoupling produced no change in the
line shape of this peak, indicating that no heteronuclear coupling is
present in this multiplet. The peak instead corresponds to H-1 signals
from 2-Man
1- residues. The unsubstituted Man
1-PO4
anomeric signal, which must be of relatively large intensity based on
the composition data in Table IV, appears to be superimposed with that
of 4-Man
1-PO4.
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DISCUSSION |
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Previous studies have shown that Leishmania
promastigotes synthesize lipid-bound (LPG), free (PG), and
protein-bound (sAP, pPPG) phosphoglycan antigens, which may play
crucial roles for virulence and transmission of this parasite life
stage (1, 3, 5, 46, 47). In the amastigote form, which causes disease
in the mammalian host, LPG expression is strongly down-regulated (23-25, 48), in most species to undetectable levels (21, 22). However,
L. mexicana amastigotes do synthesize large amounts of a
stage-specific proteophosphoglycan (aPPG) (22, 35). In this study we
have elucidated the main structural features of this novel parasite
antigen; aPPG consists of a defined polypeptide backbone, which is
modified by a variety of carbohydrate structures via Ser(P) residues.
We demonstrate that aPPG contains all glycans previously identified in
L. mexicana promastigote phosphoglycan antigens LPG and sAP
(33, 39), which include mannose, the manno-oligosaccharide series
N2a-6a and N2b, P2 and P3c (Table IV). However, the majority of the
glycans (Tables II and IV) have not been detected previously in
L. mexicana (N3b, P3b, P4c) or represent completely novel
structures (N4b, N4c, P3c', P4b', P4c', P5d, Di-P4b, Di-P4c, Di-P5b,
Di-P5d, Tri-P4c, Tri-P5b, Tri-P5c, Tri-P5d, Tri-P6b, Tri-P6c, and
Tetra-P7, Tables II and IV). Another surprising feature of aPPG is the
presence of phosphoisomers of some monophosphorylated glycans (P3c'
versus P3c and P4c' versus P4c) and the presence
of novel multiphosphorylated glycans. These glycans are phosphorylated
at the 6-position of either Gal or Glc residues, or both. To our
knowledge, Glc-6-P has not been previously observed in glycoconjugates
from any source. Neither the promastigote phosphoglycan antigens from
L. mexicana (LPG and sAP, this study; PG and
pPPG)8 nor from promastigotes
of other Leishmania species (L. major LPG, PG,
pPPG and L. donovani LPG, PG, sAP)8 contain the
novel structure elements described above. The neutral and
phosphorylated glycans are linked by phosphodiester bonds of the
conserved structure R-Man1-PO4-6-Gal-R and the newly
identified linkage R-Man
1-PO4-6-Glc-R (compare Fig.
11). Taken together, our results
suggest that the aPPG glycans could be highly branched chains as
proposed in the structure model shown in Fig. 11. This structural
arrangement is quite distinct from promastigote phosphoglycans like LPG
or sAP (compare Refs. 33 and 39). Based on the results shown in Table
IV, it can be calculated that, on average, each aPPG glycan chain
contains approximately six phosphorylated oligosaccharides, which are
capped by four neutral oligosaccharides. These glycan chains are linked
to the protein backbone most likely via the basic structure
R-Man
1-PO4-Ser (compare Fig. 11). However, the sequence,
length, and branching of the glycan chains on individual glycosylation
sites remain to be determined.
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In contrast to conventional N- and
O-glycosylation via glycosidic linkages to Asn and Ser/Thr,
respectively (49), glycosylation of proteins via phosphoamino acids is
much less common. This type of protein-glycan linkage has only been
reported in the slime mold Dictyostelium discoideum (via
Ser(P) (50)) and in the parasitic protozoa Leishmania (via
Ser(P) (33, 34)) and Trypanosoma cruzi (via Thr(P) (51)) but
appears to be absent in vertebrates. Phosphodiester linkages of the
type Man1-PO4-6-Man-R are common in cell wall
mannoproteins as well as in vacuolar and secreted enzymes of several
yeast species (52, 53 and references therein), whereas similar linkages
in glycans of vertebrates are very rare (54-56). The
phosphodiester-linked glycan structures of Leishmania aPPG
are different and far more complex. Therefore, the biosynthetic enzymes
involved in the formation of aPPG glycan structures, in particular the
phosphodiester linkages between the glycans and between Ser and the
glycan chains, could be attractive targets for the development of
specific anti-parasite drugs.
It has been shown recently that aPPG may contribute to the formation of
the huge parasitophorous vacuoles (36), which are rapidly formed after
infection of macrophages (57) and COS cells (58) by L. mexicana. In these parasite-harboring vacuoles, aPPG is present in
mg/ml concentrations (36). L. mexicana aPPG is most likely
released from the parasitophorous vacuole upon rupture of infected
macrophages and possibly also by vesicular traffic and exocytosis from
the living infected host cell (35). Released aPPG activates the
complement cascade via the lectin pathway. This unusual complement
activation by a soluble parasite product may contribute to the
pathology in the lesion (37). The structural basis for this property of
aPPG is most likely the abundance of potential mannose-binding lectin
binding sites (Man1-PO4- and (Man
1-2)1-5Man
1-PO4-, compare Table IV)
on the surface of this macromolecule. Other effects of intra- and
extracellular aPPG on the immune functions of infected mice are
currently being investigated. In particular, it appears possible that
some of the modulating activities on signal transduction, lymphokine
production, and gene expression in macrophages described for
promastigote LPG (8, 9, 12) in vitro could be mediated
in vivo in infected tissue by the structurally related
aPPG.
For aPPG biosynthesis, the expression of several novel glycosyltransferases must be activated in the amastigote. Signals from the macrophage appear to be essential for the developmental regulation of aPPG expression, since this antigen is not detected in the culture supernatant of axenic amastigotes.9 The stage-specific expression of aPPG is another example of the profound biochemical changes occurring during the L. mexicana transformation of promastigotes into amastigotes, previously documented for the expression of lysosomal cysteine proteinases (59, 60), the surface/lysosomal metalloproteinase gp63 (22, 61-63), as well as LPG and sAP (22, 46, 64). These distinctive stage-specific variations most likely reflect an adaptation to the different and more hostile environment of the phagolysosomal compartment of macrophages.
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ACKNOWLEDGEMENTS |
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We thank Dr. E. Handman for support and helpful discussions and Prof. Dr. P. Overath for suggestions on the manuscript.
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FOOTNOTES |
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* This work was supported by a fellowship of the Deutsche Forschungsgemeinschaft (to T. I.).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.
¶ To whom correspondence should be addressed: Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-72076 Tübingen, Germany. Fax: 49-7071-62971; E-mail: thomas.ilg{at}tuebingen.mpg.de.
1
The abbreviations used are: LPG,
lipophosphoglycan; PG, phosphoglycan; pPPG, promastigote
proteophosphoglycan; aPPG, amastigote proteophosphoglycan; sAP,
secreted acid phosphatase; GC-MS, combined gas chromatography-mass
spectrometry; SABG, sweet almond -glucosidase; BTBG, bovine testes
-galactosidase; AP, calf intestine alkaline phosphatase; HPAE-HPLC,
high pH anion exchange HPLC; TFA, trifluoroacetic acid; ELISA,
enzyme-linked immunosorbent assay; HF, hydrofluoric acid; ES-MS,
electrospray ionization mass spectrometry; ES-MS-MS, electrospray mass
spectrometry-mass spectrometry; TOCSY, total correlation spectroscopy;
HMBC, heteronuclear multiple bond correlation.
2 The hexose fraction from 40% HF-treated intact aPPG contained exclusively mannose, whereas the respective fraction of mild acid-treated aPPG showed 83% Man, 6% Gal, and 11% Glc. The latter treatment can lead to some limited hydrolysis of glycosidic bonds in LPG with concomitant release of pentoses and hexoses (45), whereas 40% HF seems to be more selective for phosphate esters. Therefore, it appears likely that in intact aPPG, only Man of the hexose fraction is engaged in labile phosphodiester bonds via its anomeric hydroxyl group.
3
-Glucosidase from sweet almonds cleaves
terminal
-Glc residues. It can also cleave terminal
1-3-galactosides (e.g. Gal
1-3Gal
1-4Man or
Gal
1-3Gal
1-3Gal
1-4Man) but not Gal
1-4Man (Ref. 2 and this study).
4 Gal-PO4 and Glc-PO4 from sAP and aPPG coelute in HPAE-HPLC with authentic Gal-6-PO4 and Glc-6-PO4, but since the elution behavior of Gal/Glc-2-PO4, -3-PO4, and -4-PO4 on a Carbo-Pac PA1 column is not known, a definite assignment by this method cannot be made. For the different Man-PO4, however, it has been shown that Man-6-PO4 is well separated from Man-2-PO4, -3-PO4, and -4-PO4 (65).
5 Attempts to resolve this question by reduction of P4c' and P4c with NaBD4 (2 M NH3, 1 M NaBD4, 12 h, 4 °C) were unsuccessful, because the complex ES-MS-MS fragmentation patterns obtained after reduction suggested that phosphate migration had occurred under the alkaline conditions. Similar observations were made with the diphosphorylated glycans (T. Ilg and G. Currie, unpublished results). Therefore the assignment of the sequence relies on the fact that no Man-6-PO4 was detected in aPPG (see Fig. 5). Thus the hexose at the reducing end (i.e. Man) cannot be phosphorylated.
6
-Glucosidase from sweet almonds contains low
amounts of a contaminating acid phosphatase, which converts P4c' and
P4c into N4c. N4c is then rapidly degraded to N2b by the glycosidase.
However, short term incubations of P4c result in the formation of P2,
whereas no P2 formation is observed with P4c'. Interestingly, P4c' also seems to be more resistant to the contaminating phosphatase than P4c.
7 Intramolecular phosphate migration in the gas phase may also explain the occurrence of low amounts of the "unexpected" fragment ions 241 and 259 atomic mass units in negative ion ES-MS-MS of P4b and P5b of L. major LPG, P3c of L. mexicana LPG and sAP, P3c and P4c of L. mexicana aPPG (Table III and Fig. 7), as well as the low amount of unexpected ions identified in all other samples investigated in this study (Table III and Fig. 7).
8 T. Ilg, unpublished results.
9 T. Ilg and A. Aebischer, unpublished observations.
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REFERENCES |
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