(Received for publication, May 17, 1996, and in revised form, October 24, 1996)
From the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007
The nature and extent of carbohydrate modification in intraerythrocytic stage Plasmodium falciparum proteins have been controversial. This study describes the characterization of the carbohydrates in intraerythrocytic P. falciparum proteins and provides an overall picture of the nature of carbohydrate modification in the parasite proteins. P. falciparum strains were metabolically labeled with radioactive sugar precursors and ethanolamine at different developmental stages. The individual parasite proteins separated on SDS-polyacrylamide gels and whole parasite cell lysates were analyzed for the carbohydrate moieties. The results established the following: 1) glycosylphosphatidylinositol (GPI) anchors represent the major carbohydrate modification in the intraerythrocytic stage P. falciparum proteins; 2) in contrast to previous reports, O-linked carbohydrates are either absent or present only at very low levels in the parasite; and 3) P. falciparum contains low levels of N-glycosylation capability. The amount of N-linked carbohydrates in whole parasite proteins is ~6% compared with the GPI anchors attached to proteins based on radioactive GlcN incorporated into the proteins.
The glycan cores of multiple parasite protein GPI anchors are all
similar, consisting of
protein-ethanolamine-phosphate-(Man1-2)6Man
1-2Man
1-6Man
1- 4GlcN.
The fourth Man residues distal to GlcN of the GPI anchor glycan
cores contain unidentified substituents that are susceptible to
conditions of nitrous acid deamination. This unusual structural feature
may contribute to the reported pathogenic properties of the P. falciparum GPI anchors.
Malaria, a life-threatening disease caused by parasitic protozoa of the genus Plasmodium, is a major health problem throughout the tropical and subtropical regions of the world. Among the four species that infect humans, Plasmodium falciparum is the most virulent. New approaches such as vaccine development and novel therapeutic agents are urgently needed due to the emergence of parasite strains resistant to chloroquine and other commonly used drugs (1).
Vaccines based on antigens of the blood stage parasite are under intensive study. A major focus has been on synthetic peptides or recombinant proteins of cell surface antigens (2-6). However, this approach has not been highly effective, although immunization with native cell surface proteins purified from the erythrocytic stage parasite is known to confer significant protective immunity (7-10). It is plausible that post-translational modifications play an important role in antigenicity. Accordingly, a basic understanding of such modifications may assist in the development of effective vaccines.
Glycosylation is an often extensive post-translational modification of eukaryotic proteins. Carbohydrate moieties of glycoproteins perform a variety of functions including modulation of immunological properties, receptor-ligand interactions, sorting and localization of proteins, cell adhesion, and cell-cell communication (11). In addition, they contribute to protein conformation and, thus, to proteolytic processing (11). Finally, the carbohydrate moieties can be highly antigenic and may contribute to disease pathology.
Several proteins of erythrocytic stage P. falciparum are known to contain carbohydrates (12-23). The deduced amino acid sequences of the parasite proteins have potential N-glycosylation sites (7, 24-26). However, Dieckmann-Schuppert et al. (27) have reported that P. falciparum has no N-glycosylation capability based on their finding that dolichol phosphate-linked oligosaccharides, the obligatory biosynthetic intermediates, and dolichol phosphate-oligosaccharyltransferase activity were not detectable in the parasite. However, while this work was in review, Kimura et al. (28) reported the existence of N-linked glycans in P. falciparum.
Two laboratories have previously reported that O-glycosylation is the major carbohydrate modification in proteins of the intraerythrocytic asexual stage of P. falciparum (21, 23) and that the O-linked carbohydrates are mainly single residues of GlcNAc with the remainder being oligosaccharides, some containing GlcNAc at the reducing end. While one group reported that the parasite proteins also contain O-linked GalNAc (19, 21), another group showed the absence of this sugar moiety and the lack of its biosynthesis (23). Kimura et al. reported the presence of O-glycanase-releasable oligosaccharides as the major carbohydrates in the proteins of late trophozoite and schizont stage P. falciparum (28).
Previous studies reported the presence of glycosylphosphatidylinositol
(GPI)1 in several proteins of erythrocytic stage P. falciparum including merozoite surface protein 1 (MSP-1), merozoite surface protein 2 (MSP-2), 72-kDa heat shock protein
(HSP-72), 102-kDa transferrin receptor, and 75-kDa serine protease
(29-32). Recently, Gerold et al. (33) studied the GPI
lipids (not anchored to proteins) of P. falciparum. Two
putative GPI anchor precursors,
ethanolamine-phosphate-(Man1-2)6Man
1-2Man
1-6Man
1-4GlcN-PI and ethanolamine-phosphate-6Man
1-2Man
1-6Man
1-4GlcN-PI, were identified (33). While the current study was in progress, Gerold et al. (34) reported the structures of the GPI anchors of
P. falciparum MSP-1 and MSP-2. Both were shown to
consist of
ethanolamine-phosphate-(Man
1-2)6Man
1-2Man
1-6Man
1-4Glc4GlcN1-phosphatidylinositol,
bearing a myristic acid substitution on the inositol ring and
predominantly palmitic acid in the diacylglycerol moiety (34).
In this study, using several P. falciparum strains, we clearly demonstrate that O-linked carbohydrates are either absent or present only at very low levels in intraerythrocytic P. falciparum proteins. In agreement with Kimura et al. (28), we show that P. falciparum apparently contains N-linked carbohydrates. However, in contrast to the former study (23), our results establish that N-linked oligosaccharides are minor constituents, and that GPI anchors represent the major carbohydrate modification in intraerythrocytic P. falciparum proteins. Furthermore, we establish that GPI anchors of the parasite proteins have Man4-GlcN cores with substituents that are susceptible to the conditions of nitrous acid deamination on the terminal Man residues. These unusual structures may contribute to the reported cellular and immunological functions of P. falciparum GPI anchors (35, 36).
Aspergillus saitoi -mannosidase
(400 milliunits/mg), jack bean
-mannosidase (30 units/mg), bovine
milk GalT, and a mixture of Glc oligomers were from Oxford Glycosystems
(Rosedale, NY); PMSF and (4-amidinophenyl)-methylsulfonyl fluoride
hydrochloride were from Boehringer Mannheim; N-glycanase was
from New England Biolabs (Beverly, MA);
[1-3H]ethanolamine hydrochloride (10-30 mmol),
L-[6-3H]Fuc (60-90 Ci/mmol),
D-[6-3H]GlcN hydrochloride (40-60 Ci/mmol),
D-[6-3H]Gal (40-60 Ci/mmol),
D-[6-3H]Man (20-30 Ci/mmol),
UDP-[6-3H]Gal (40-60 Ci/mmol), and
UDP-[6-3H]GlcNAc (40-60/mmol) were from American
Radiolabeled Chemicals (St. Louis, MO); rainbow protein molecular
weight markers, rainbow 14C-methylated protein molecular
weight markers, and AmplifyTM fluorographic solution were
from Amersham Corp.; Bio-Gel P-4 (fine and extra fine), AG 50W-X12
(H+), AG 50W-X16 (H+), and AG 4-X4 (base) were
from Bio-Rad; HPTLC plates coated with silica gel 60 on glass plates
and aqueous HF (52%) were from EM Science (Gibbstown, NJ); chicken
ovalbumin (grade V), leupeptin, pepstatin A, chymostatin, antipain,
TLCK, TPCK, AMP, benzamidine, iodoacetamide, and sodium borohydride
were from Sigma; EN3HANCETM
fluorographic spray was from DuPont NEN; Percoll was from Pharmacia Biotech Inc.; human erythrocytes (blood group O) were from the American
Red Cross (Baltimore, MD); human serum was from Interstate Blood Bank
Inc. (Memphis, TN); RPMI 1640 medium was from Life Technologies, Inc.
Man
1-2Man
1-2Man
1-6Man
1-4AHM (Man4-AHM) and
a mixture containing 2,5-anhydromannitol (AHM), Man
1-4AHM (Man-AHM), Man
1-6Man
1-4AHM (Man2-AHM),
Man
1-2Man
1-6Man
1-4AHM (Man3-AHM) and
Man4-AHM were a generous gift from Dr. Alvaro A. Serrano
and were derived from the GPI anchors of mucin-like glycoproteins of
Trypanosoma cruzi (37).
Asexual blood stage P. falciparum parasite clone FCR-3 (obtained from Gambian isolates) was provided by Dr. Isabella Quakyi (Department of Biology, Georgetown University). Clone D6 (isolated from Sierra Leone I/CDC isolate) and clone W2 (from a 50:50 mixed culture of Indochina III/CDC and Sierra Leone I/CDC isolates) (38) were provided by Dr. Dennis E. Dyke (Walter Reed Army Institute of Research, Washington, D. C.). Parasite strain NF54 (isolated from a patient in Amsterdam infected with an African strain; Ref. 39) was provided by Dr. Stephen Hoffman (Naval Medical Research Institute, Bethesda, MD).
Parasite strains were maintained as asynchronous continuous cultures in RPMI 1640 medium supplemented with 25 mM HEPES, 25 mM sodium bicarbonate, 0.5% hypoxanthine, and 10% human serum at 3-4% hematocrit. Cultures were incubated at 37 °C in an atmosphere of 90% N2, 5% O2, and 5% CO2. Parasite growth was monitored by counting the infected erythrocytes in Giemsa-stained thin blood smears under light microscopy.
Metabolic Labeling of Parasite Proteins with Radioactive PrecursorsParasite cultures at 12-14% parasitemia were synchronized to the ring stage by treating with 5% sorbitol for 5 min (40) and then washed and suspended in complete medium. Parasitemia was adjusted to 10% by the addition of fresh erythrocytes, and the cells were incubated at 3% hematocrit for 16-18 h. The parasites were then metabolically labeled with 3H-sugars (50 µCi/ml) in medium (10 ml) containing 5 mM D-Glc and 10% human serum for 6-8 h. To label the parasites at different developmental stages, cultures were treated with [3H]GlcN for 16 h after synchronization with sorbitol and for 6-8 h each after the synchronized cultures were maintained in complete medium for 18, 24, 30, and 36 h. Labeling with [3H]ethanolamine (50 µCi/ml) was carried out in complete medium.
Isolation of Parasite-infected Erythrocytes and Preparation of Cell LysateAfter metabolic labeling, erythrocytes were harvested
by centrifugation at 1500 × g and washed with RPMI
1640 medium. The cells were suspended in RPMI 1640 medium, layered onto
70% isotonic Percoll, and centrifuged at 2700 × g for
20 min (41). The infected erythrocytes in the Percoll layer were
recovered, washed, and then treated with 50 mM Tris-HCl,
100 mM NaCl, 5 mM EDTA, pH 8.0, containing 2%
Nonidet P-40, 1 mM (4-amidinophenyl)-methylsulfonyl fluoride hydrochloride, 0.2 mM leupeptin, 0.2 mM chymostatin, 0.2 mM TPCK, 0.2 mM
TLCK (infected erythrocyte pellet:lysis buffer, 1:20 (v/v)) at 4 °C
for 20 min. The cell lysates (Percoll-enriched infected erythrocytes
from 10 ml of culture) were centrifuged at 3500 × g at
4 °C for 10 min and then at 100,000 × g at 4 °C for 30 min. The supernatants were concentrated to 200-250 µl in a
Speed-Vac and stored at 70 °C.
SDS-PAGE (42) was performed with 5-20%, 1.5-mm-thick,
polyacrylamide gradient gels. The 3H-labeled parasite cell
lysates were mixed with an equal volume of 125 mM Tris-HCl,
pH 6.8, containing 4% SDS and 20% glycerol, heated in a boiling water
bath for 5 min, and electrophoresed. After the run, the gels were
treated with MeOH, water, glacial HOAc (50:40:10, v/v/v) for 1 h,
washed with water for 5 min, and soaked in AmplifyTM
fluorographic solution for 1 h. The gels were dried under vacuum at 65 °C and exposed to x-ray film at 70 °C.
The 3H-labeled parasite protein bands in the SDS-polyacrylamide gels, visualized by salicylic acid-enhanced fluorography (43), were excised, and the filter paper backing was scraped off. The procedure outlined here is for a single protein band. For large scale isolation, corresponding protein bands from different lanes were combined, and reagents were appropriately scaled up. The gels were cut into ~1-mm pieces and suspended in water (1 ml). The excess water was removed, and the gel was washed with water (3 × 1 ml) to remove the radioactivity enhancer (44). In some instances, water-swollen gel pieces were washed with MeOH (3 × 1 ml), which also removes the enhancer effectively. The water-swollen gel pieces were treated with an equal volume of 100 mM NaOH, 1 M NaBH4, diluted to 2 ml with 50 mM NaOH, 0.5 M NaBH4, and incubated at 44 °C for 24 h (45). The reaction mixture was cooled in an ice bath and neutralized with 2 M HOAc, and the clear solution was removed. The gel pieces were washed with water (5 × 2 ml) and then with MeOH (3 × 2 ml). The combined reaction solution and washes were centrifuged at 10,000 × g to remove insoluble particles. The supernatant was then dried in a rotary evaporator at 35 °C. Boric acid was removed by evaporation with 0.1% HOAc in MeOH (3 × 4 ml). The residue was dissolved in water and chromatographed on Bio-Gel P-4 (see below). Typically 20,000-30,000 cpm of [3H]GlcN-labeled carbohydrates were recovered from the 200-215-, 82-, and 75-kDa protein bands.
Isolation of Intact GPI Anchor of Parasite Proteins Separated on Polyacrylamide GelsThe protein bands in the SDS-polyacrylamide gels were cut into small pieces, washed with water and MeOH, and then dried in a Speed-Vac. The gels were suspended in water (0.5 ml) and homogenized to a fine paste using a glass minihomogenizer. The pastes (from two bands of the same protein) were suspended in 4 ml of 100 mM Tris-HCl, 1 mM CaCl2, pH 8.0, containing 0.05% SDS and 0.5% Nonidet P-40, and incubated with Pronase (5 mg; 1-mg aliquots were added at intervals of 8-12 h) at 55 °C for 60 h. The enzyme digests were centrifuged, and the gels were washed with water (3 × 2 ml) and then with MeOH (2 × 2 ml). The combined supernatants and washings were dried in a rotary evaporator at 30 °C. The residues were dissolved in water (0.5 ml) and then either chromatographed on Bio-Gel P-4 (1 × 90 cm) in 100 mM pyridine, 100 mM HOAc, pH 5.2, or extracted with water-saturated 1-butanol (4 × 0.5 ml) (46-49). The 1-butanol layers were extracted with water (2 × 0.5 ml), and the organic layers containing the GPI anchor were dried. About 20,000-26,000 cpm of GPI anchors were obtained from a major [3H]GlcN-labeled protein band.
Preparation of Delipidated P. falciparum Cell LysatesThe
parasite cultures at early and late trophozoite stages were labeled
with [3H]GlcN for 10-18 h, and the infected erythrocytes
were enriched by centrifugation on a Percoll cushion as described
above. The cells were extracted three times with CHCl3,
MeOH, water (10:10:3, v/v/v) to remove glycolipids including free
GPI-anchors. The pellet was dissolved in 25 mM Tris-HCl, pH
7.5, containing 1% SDS, 10 mM benzamidine, 5 mM EDTA, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 2 µg/ml antipain, and 1 µg/ml chymostatin, and
dialyzed (Mr cut-off of 6,000-8,000)
extensively for 48 h against the above buffer followed by dialysis
for 1 h against distilled water containing protease inhibitors.
The lysates were lyophilized, washed with 90% MeOH, and then
subjected to 1) alkaline -elimination, 2) digestion with Pronase, or
3) digestion with N-glycanase.
The parasite cell lysate (~120,000 cpm) was dissolved in 50 mM NaOH, 0.5 M NaBH4 (2 ml), incubated at 44 °C for 22 h, and then neutralized with 2 M HOAc in an ice bath. The solution was dried, boric acid was removed with MeOH, the residue was dissolved in water, and then the solution was chromatographed on Bio-Gel P-4 (1 × 90 cm) in 0.1 M pyridine, 0.1 M HOAc, pH 5.2. Fractions containing radioactivity were pooled and lyophilized.
Digestion of P. falciparum Cell Lysate with N-GlycanaseThe
[3H]GlcN-labeled parasite lysate (~110,000 cpm) was
suspended in 300 µl of 0.5% SDS, 1% -mercaptoethanol and then
heated in a boiling water bath for 10 min and cooled. Then 0.5 M sodium phosphate, pH 7.5 (35 µl), and 10% Nonidet P-40
(35 µl) were added. The suspension was sonicated in an ultrasonic
bath for 10 min to obtain a clear solution, and then
N-glycanase (400 IUB milliunits) was added and incubated at
37 °C for 36 h. Four volumes of MeOH were added, and the
solution was chilled on dry ice for 10 min and then centrifuged. The
supernatant was dried in a Speed-Vac, and the residue was dissolved in
water and extracted with water-saturated 1-butanol to remove the
detergents. The aqueous phase was chromatographed on Bio-Gel P-4
(1 × 90 cm) in 0.1 M pyridine, 0.1 M
HOAc, pH 5.2. The radioactive fractions were pooled and lyophilized,
and their carbohydrate compositions were determined before and after
reduction with NaBH4.
The [3H]GlcN-labeled parasite lysate (~110,000 cpm) was suspended in 100 mM Tris-HCl, 1 mM CaCl2, pH 8.0 (0.5 ml) and incubated with Pronase (4 × 1 mg, added at 10-12-h intervals) at 37 °C for 48 h. The solution was extracted with water-saturated 1-butanol (3 × 0.5 ml), and the two phases were separately dried in a Speed-Vac. The residue from the 1-butanol phase was analyzed for radiolabeled carbohydrates by HPAEC before and after treatment with HN02. The residue from the aqueous phase was dissolved in water and chromatographed on Bio-Gel P-4 (1 × 90 cm) in 0.1 M pyridine, 0.1 M HOAc, pH 5.2. The eluted radioactive components were analyzed for carbohydrates.
Analysis of P. falciparum Proteins for Terminal GlcNAc Residues by GalactosylationErythrocytes harboring trophozoite and schizont stage parasites were enriched by centrifugation on a Percoll cushion and lysed with 0.015% saponin (18). The released parasites were washed with PBS and then lysed with 25 mM HEPES, pH 7.3, containing 0.05% SDS, 1% Nonidet P-40, 0.1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml chymostatin, 2 µg/ml antipain, and 5 mM benzamidine. To the parasite cell lysate containing 100 µg of protein was added 20 µl of 100 mM HEPES, pH 7.3, containing 100 mM Gal, and 50 mM MnCl2, 100 milliunits of GalT, 2 µl of 100 × protease inhibitor stock solutions. The solution was diluted with water (180 µl), 5 µCi of UDP-[3H]Gal in water (20 µl) containing 25 mM AMP was added, and the solution was incubated at 37 °C for 2 h (50). The reaction was stopped by the addition of 100 mM EDTA, 10% SDS (20 µl). Aliquots of the reaction mixture containing 30-40 µg of parasite proteins were analyzed by SDS-PAGE fluorography using a 7-20% SDS-polyacrylamide gradient gel (42).
Crude erythrocyte ghosts were prepared by the lysis of human red cells with 1:20 diluted phosphate-buffered saline for 30 min, centrifuged, and washed with the same buffer. The pellet was suspended in 25 mM HEPES, 1% Nonidet P-40, pH 7.3. The detergent-solubilized human erythrocyte ghosts and ovalbumin were galactosylated in parallel as positive controls and analyzed by SDS-PAGE fluorography.
Analysis of P. falciparum Cell Lysate for Peptide O-Glycosidic GlcNAcTThe analysis was performed as described by
Dieckmann-Schuppert et al. (23). The saponin-released
parasites were lysed in ice-cold water containing 1 µg/ml leupeptin,
2 µg/ml antipain, 1 µg/ml pepstatin A, 1 µg/ml chymostatin, 1 mM PMSF, and 5 mM iodoacetamide. To the lysate
was added an equal volume of 25 mM Tris-HCl, pH 7.2, containing 5 mM MgCl2, 5 mM
MnCl2, 0.8% Triton X-100. The parasite cell lysate (200 µl) corresponding to 108 parasites was incubated with
2-5 µCi of UDP-[3H]GlcNAc and 1 mM
synthetic peptide Pro-Tyr-Thr-Val-Val at 37 °C for 30-60 min.
Control incubations not containing the synthetic peptide were carried
out in parallel. The reaction mixture was diluted with 0.9 ml of
ice-cold water and then deionized on a column of Ag 1-X 8 (Cl). The effluents and washings were concentrated,
extracted with 1-butanol to remove the detergent, and then
chromatographed on Bio-Gel P-2 (0.9 × 50 cm) in 0.1 M
pyridine, 0.1 M HOAc, pH 5.2. Fractions (0.5 ml) were
collected, and the radioactivity was measured by liquid scintillation
counting.
The peptide Pro-Tyr-Thr-Val-Val was custom synthesized by Research Genetics (Huntsville, AL). The crude peptide was over 95% pure by HPLC and mass spectral analysis; it was further purified by gel filtration on Bio-Gel P-2 (0.9 × 50 cm). Fast atom bombardment mass spectrometry analysis indicated that the purified peptide was over 98% pure.
Gel Filtration ChromatographyGel filtration of radioactive components released from proteins by Pronase digestion or treatment with NaOH/NaBH4 was performed on columns (1 × 90 cm) of Bio-Gel P-4 (fine) equilibrated with 100 mM pyridine, 100 mM HOAc, pH 5.2. Fractions (1.1 ml) were collected, and aliquots were assessed for 3H by liquid scintillation counting. The glycan cores, isolated by treating the parasite protein GPI anchors with 50% aqueous HF, HNO2, and NaBH4, were similarly chromatographed. Fractions containing radiolabeled carbohydrates were pooled, lyophilized, and deionized with AG 50W-X12 (H+) and AG 4-X4 (base).
Mild Alkaline Hydrolysis of GPI AnchorThe GPI anchors, isolated by Pronase digestion (20,000-30,000 cpm) of protein gel bands, were treated with 100 µl of 25 mM NaOH overnight at 37 °C. The solutions were neutralized with an equal volume of 100 mM HOAc and analyzed on columns (1 × 90 cm) of Bio-Gel P-4 in 100 mM pyridine, 100 mM HOAc, pH 5.2.
N-Acetylation (46, 47)The [3H]GlcN-labeled GPI anchors (~10,000 cpm) were dissolved in 100 µl of aqueous sodium bicarbonate (100 mg/ml) and cooled in an ice bath. Acetic anhydride (4.5 µl) was added in three equal aliquots at 10-min intervals, and the solutions were warmed to room temperature. After 30 min, the reaction mixtures were deionized with AG 50W-X16 (H+) and then dried in a rotary evaporator.
Periodate OxidationThe [3H]GlcN-labeled GPI anchors obtained by the Pronase digestion and extraction with 1-butanol were treated, before and after N-acetylation, with 25 mM sodium periodate in 50 mM NaOAc, pH 5.5 (200 µl) for 16 h at room temperature in the dark. Excess periodate was destroyed by the addition of glycerol (10 µl), and the products were chromatographed on a column of Bio-Gel P-4 in 100 mM pyridine, 100 mM HOAc, pH 5.2. Fractions containing radioactivity were pooled, lyophilized, and then hydrolyzed with 4 M HCl for hexosamine analysis.
Dephosphorylation with Aqueous HFThe 3H-labeled GPI anchors (30,000-40,000 cpm), isolated from protein bands either by NaOH/NaBH4 treatment or digestion with Pronase, were treated with 50% aqueous HF (50 µl) in an ice bath for 48 h (46-49). The acid was neutralized with frozen saturated LiOH and centrifuged, and the supernatant was chromatographed on Bio-Gel P-4 columns (1 × 90 cm) in 100 mM pyridine, 100 mM HOAc, pH 5.2.
Deamination with HNO2The [3H]GlcN-labeled GPI anchors (30,000-150,000 cpm), isolated from protein bands either by NaOH/NaBH4 treatment or digestion with Pronase, were treated with 0.2 M NaOAc, pH 3.8 (75 µl) and 1 M NaNO2 (75 µl) (46-49). After 18 h at room temperature, saturated boric acid (60 µl) was added, and the pH of the solution was adjusted to 10-11 with 2 M NaOH. Immediately, 1 M NaBH4 in 100 mM NaOH (100 µl) was added and allowed to react at room temperature for 5-6 h. Excess NaBH4 was destroyed by acidifying to pH 5 with cold 2 M HOAc in an ice bath, the solution was dried in a rotary evaporator, and boric acid was removed by repeated evaporation with MeOH. The samples were chromatographed on columns of Bio-Gel P-4 (1 × 90 cm) in 100 mM pyridine, 100 mM HOAc, pH 5.2.
Isolation of GPI Glycan CoreThe GPI anchors (30,000-40,000 cpm) isolated from gel bands were first dephosphorylated and then deaminated as above (46-49). The samples were desalted using AG 50W-X16 (H+) and AG 4-X4 (base) resins.
Partial Acetolysis of the GlycansThe GPI anchor glycan cores from 200-215- and 82-kDa proteins (20,000-30,000 cpm) isolated as above were dried in reactive vials and peracetylated with 40 µl of pyridine, acetic anhydride (1:1, v/v) at room temperature for 18 h (47-49). The solutions were dried in a Speed-Vac and then treated with 30 µl of acetic anhydride, HOAc, concentrated sulfuric acid (10:10:1, v/v/v) for 8 h at 37 °C (47-49). To each reaction mixture, 10 µl of pyridine and 500 µl of water were added and then extracted with CHCl3 (250 µl). The organic layers were washed with water, dried, and treated with 30% ammonia (200 µl), MeOH (1:1, v/v) at 37 °C for 24 h. The solutions were dried in a Speed-Vac, and the residues were dissolved in water and then chromatographed on Bio-Gel P-4 in 100 mM pyridine, 100 mM HOAc, pH 5.2.
Partial HydrolysisThe [3H]GlcN-labeled GPI
anchors (60,000-100,000 cpm) isolated by NaOH/NaBH4
treatment of 200-215- and 82-kDa protein bands were deaminated with
HNO2 and reduced with NaBH4. The samples were
dissolved in 0.1 M trifluoroacetic acid (400 µl), heated at 100 °C for 4 h (46, 49), and then dried in a Speed-Vac. About half of the partial hydrolysates were treated with 25 µl (30 units/ml) of jack bean -mannosidase, deionized with AG 50W-X16 (H+) and then dried. The enzyme-treated and untreated
partial hydrolysates were dephosphorylated with 50% aqueous HF and
then deionized with AG 50W-X16 (H+) and AG 4-X4 (base)
resins.
For hexosamines and hexosaminitols analysis, the [3H]GlcN-labeled parasite carbohydrate moieties (2000-4000 cpm) were hydrolyzed with 3 M HCl at 100 °C for 6 h and then dried in a Speed-Vac. For sialic acid analysis, samples were hydrolyzed with 50 mM sulfuric acid at 80 °C for 50 min, neutralized with 0.2 M NaOH. The hydrolysates were mixed with appropriate standard sugars and analyzed with a Dionex BioLC HPLC system coupled to pulsed amperometric detection using a CarboPac PA1 high pH anion exchange column (4 × 250 mm) or a CarboPac MA1 high pH anion exchange column (4 × 250 mm) (51). The eluents were as follows: 1) 20 mM NaOH at a flow rate of 0.8 ml/min, for hexosamines on CarboPac PA1; 2) 100 mM NaOH, 150 mM NaOAc at a flow rate of 0.8 ml/min, for sialic acids on CarboPac PA1; and 3) 100 mM NaOH for 10 min and then a gradient elution to increase the concentration of NaOH to 600 mM over a period of 10 min (flow rate, 0.4 ml/min), for hexosaminitols on CarboPac MA1. Elution of radioactive sugars was monitored by liquid scintillation counting. 3H-Sugars in samples were identified by either coelution or comparison of elution time with standard sugars. Nonradioactive standard sugars were detected by pulsed amperometric detection.
Size Analysis of Glycans by Bio-Gel P-4 ChromatographyThe neutral glycans of the parasite GPI anchors, their partial hydrolysates, and the products of exoglycosidase digestion and partial acetolysis were analyzed on columns of Bio-Gel P-4 (extra fine, 1 × 115 cm) in water at 60 °C at a flow rate of 2 ml/h (46-48, 52). Fractions (0.5 ml) were collected, and radioactivity was measured by liquid scintillation counting. The column was calibrated with 3H-labeled Glc oligomers, standard Man4-AHM, and a mixture containing AHM, Man-AHM, Man2-AHM, Man3-AHM, and Man4-AHM. The GPI glycans were characterized by size comparison with Glc oligomers and by comparison of retention times with those of standards. Glc was routinely used as an internal standard, and the elution was monitored with the phenol sulfuric acid reagent.
Analysis of GPI Glycans by High pH Anion Exchange ChromatographyThe neutral glycans of the GPI anchors and the products of exoglycosidase digestion were also analyzed on CarboPac PA1 (4 × 250 mm) using the Dionex HPLC system (46-48). Elution was with 100% of 100 mM NaOH and 0% of 100 mM NaOH, 250 mM NaOAc at sample injection and then a linear gradient to 40% of 100 mM NaOH, 250 mM NaOAc at 35 min and then maintenance of 40% of 100 mM NaOH, 250 mM NaOAc for 10 min. Fractions (0.4 ml) were collected, and the radioactivity was measured. Glc oligomers were added to samples as internal standards, and were monitored by pulsed amperometric detection. The [3H]GlcN-labeled parasite glycans were identified by comparison of elution time with those of Glc oligomers and standard AHM, Man-AHM, Man2-AHM, Man3-AHM, and Man4-AHM.
TLC Analysis of GPI AnchorsThe glycan cores of the
parasite GPI anchors, their partial hydrolysates, and the products of
exoglycosidase digestion and partial acetolysis were also analyzed by
HPTLC (46, 49). The plates were developed with 1-propanol, acetone,
water (9:6:4, v/v/v), dried, sprayed with EN3HANCE, and
then exposed to x-ray film at 70 °C. The parasite GPI glycans were
identified by comparison of retention times with those of
Man4-AHM standard and an AHM to Man4-AHM
ladder.
GPI glycans were
treated with 25-50 µl of jack bean -mannosidase (30 units/ml) in
100 mM NaOAc, pH 5.0, containing 2 mM
Zn2+ at room temperature for 2 h and then at 37 °C
for 22 h (46, 47, 49). The solutions were heated in a boiling
water bath for 5 min and desalted either with AG 50W-X16
(H+) resin or by gel filtration on Bio-Gel P-4 columns
(1 × 90 cm) in 100 mM pyridine, 100 mM
HOAc, pH 5.2.
The GPI glycans
were incubated with 20 µl of A. saitoi -mannosidase (1 milliunit/ml) in 100 mM NaOAc, pH 5.0, at 37 °C for 22 h (46, 47, 49). The solutions were heated in a boiling water
bath for 5 min and desalted with AG 50W-X16 (H+) and AG
4-X4 (base) resins.
Intraerythrocytic stage P. falciparum was
metabolically labeled with [3H]GlcN,
[3H]Man, [3H]Gal, or [3H]Fuc
in medium containing 5 mM D-Glc and 10% human
serum. SDS-PAGE analysis of the parasitized erythrocyte cell lysates
and fluorographic identification of the protein bands demonstrate that
[3H]GlcN was incorporated into more than 15 proteins
(size range, 14-260 kDa) (Fig. 1).
[3H]Man gave a similar labeling pattern, but the
incorporation of radioactivity was only about 5-10% of that observed
with [3H]GlcN (data not shown). [3H]Gal and
[3H]Fuc were not significantly incorporated into the
parasite proteins (data not shown). Since the incorporation of
[3H]Man to the parasite proteins was low, subsequent
metabolic labeling was routinely performed with
[3H]GlcN.
Metabolic labeling with [3H]GlcN was performed at different developmental stages of the parasite (rings undergoing transformation to schizonts) (Fig. 1). Among the radiolabeled parasite proteins (Fig. 1A), 200-215-, 82-, and 75-kDa proteins were predominant. Two proteins (36 and 53 kDa) were labeled relatively intensely by the ring stage parasite, and their intensity drastically decreased in the trophozoites and schizonts. The incorporation of [3H]GlcN by the schizonts was significantly lower (Fig. 1A, lanes 6 and 7) compared with the rings and trophozoites (Fig. 1A, lanes 1-5). At least four radiolabeled proteins (38, 43, 46, and 56 kDa, some not well resolved on the gel) appear to be synthesized only at the trophozoite and schizont stages (Fig. 1A, compare lanes 2-7 with lane 1). A 260-kDa protein band was labeled to a significant level only by the ring stage parasites (Fig. 1A, lane 1). Several minor proteins with molecular weights ranging from 130,000 to 200,000 were also labeled with [3H]GlcN. In contrast to a previous report (23), noninfected red cells did not incorporate radiolabeled precursors (Fig. 1B, lane 4); >99% of the incorporated radioactivity was parasite-dependent. Previously, Udeinya and Van Dyke (18) and others (28) reported that noninfected erythrocytes do not incorporate [3H]GlcN into proteins.
To determine whether the carbohydrate modification of P. falciparum proteins differs between strains, three other parasite strains, W2, D6, and NF54, were also metabolically labeled with [3H]GlcN. SDS-PAGE of the cell lysates and fluorography revealed similar protein labeling patterns to that observed with the FCR-3 strain, with the exception of the altered mobility of the 75- and 82-kDa proteins (Fig. 1, compare panel A, lane 1, with panel B). In the W2 strain, the mobility of these two proteins was slower than in the FCR-3 strain, and they electrophoresed as two distinct, widely separated bands (Fig. 1B, lane 2). In the D6 strain, the mobility of the 75- and 82-kDa proteins (Fig. 1B, lane 1) was comparable with the respective proteins of the FCR-3 strain. These proteins appear as a single band in the NF54 strain (Fig. 1B, lane 3). The difference in mobility of 75- and 82-kDa proteins from different parasite strains is not due to altered glycosylation, since they have similar carbohydrate moieties (see below).
Analysis of SDS-PAGE-separated P. falciparum Proteins for Carbohydrate MoietiesAfter SDS-PAGE of the parasite lysates and
fluorography, the individual [3H]GlcN-labeled protein gel
bands were separately excised and washed with water to remove the
radioactive enhancer. More than 98% of the radiolabeled proteins
remained in the gel slices. Pronase digestion (200-215-, 82-, and
75-kDa proteins from the FCR-3 strain) or alkaline borohydride
treatment (all radiolabeled proteins from four parasite strains)
released more than 95% of the radiolabeled carbohydrate moieties from
the gel slices. These were isolated by gel filtration using Bio-Gel P-4
(Figs. 2 and 3). Approximately 95% of
the Pronase-released carbohydrate eluted in the void volume along with
the detergents from the reaction mixture, and the remainder was eluted
at a 1200-1600-Da range (Fig. 2). However, after treatment with mild
alkali or HNO2, all of the radioactivity was eluted at a
1200-1600-Da range (Fig. 2).
The radiolabeled carbohydrates released by NaOH/NaBH4
treatment of 200-215-, 43-46-, and 14-kDa protein bands eluted on
Bio-Gel P-4 as single symmetrical peaks corresponding to a molecular
weight of 1200-1600 (Fig. 3). In contrast, the radiolabeled
carbohydrates from the 36-38-, 53-56-, 75-, and 82-kDa protein bands
(FCR-3 strain) eluted as broad (in some cases heterogeneous) peaks at a
1200-2400-Da range (Fig. 3C). Upon digestion with Pronase,
however, all of the samples were eluted as symmetrical peaks at a
molecular weight range similar to that from the 200-215-kDa protein
(Fig. 3D), suggesting that the observed higher sizes were
due to peptide moieties associated with the carbohydrates. Therefore,
the release of radiolabeled carbohydrates from parasite proteins by
NaOH/NaBH4 is not due to classical -elimination of
Ser/Thr-linked O-glycosidic bonds but apparently to alkaline
degradation of protein moieties. The [3H]GlcN-labeled
peptide-carbohydrate moieties obtained from various proteins of the D6,
W2, and NF54 strains were also analyzed (data not shown). In all cases,
the results were similar to those from the FCR-3 strain. In no case did
significant radioactivity elute at the hydrodynamic volume
corresponding to hexosamines or hexosaminitols.
The [3H]GlcN-labeled carbohydrate moieties released by Pronase digestion of the parasite protein bands (200-215, 82, and 75 kDa from the FCR-3 strain at different erythrocytic stages) were partitioned between water and water-saturated 1-butanol. Approximately 95% of the radioactivity was extracted into the organic phase, indicating that the carbohydrate moieties contain hydrophobic substituents. However, after mild saponification, the Pronase-released carbohydrates remained in the aqueous phase, suggesting that the hydrophobic moieties are acyl esters. Further, the carbohydrate moieties released from the protein bands (by treatment with Pronase or alkaline borohydride) bind almost quantitatively to anion exchange resin (data not shown). This binding was abolished upon treatment with 50% aqueous HF, which removes phosphate ester groups. Thus, phosphate ester group(s) are presumed to be present. The parasite carbohydrate moieties also quantitatively bind to a cation exchange resin. This binding was abolished upon N-acetylation or deamination with HNO2, indicating the presence of free amino groups.
Dionex-HPAEC analysis of the acid hydrolysates of the carbohydrates (Table I), isolated from the [3H]GlcN-labeled 200-215-, 82-, and 75-kDa parasite proteins (separately from each of the FCR-3, D6, W2, and NF54 strains) after Pronase digestion, showed the presence of GlcN but neither sialic acid nor galactosamine. Compositional analysis of the carbohydrate moieties isolated by alkaline borohydride treatment of the major [3H]GlcN-labeled parasite proteins (from the FCR-3, D6, W2, and NF54 strains) also gave only GlcN; glucosaminitol and galactosaminitol were not detected (Table I). In contrast to previous reports (19-23, 28), these results demonstrate that the previously reported O-linked carbohydrates are either absent or present at very low levels in the parasite proteins.
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The [3H]GlcN-labeled carbohydrate moieties of the parasite proteins (14, 36-38, 43-46, 53-56, 75, 82, and 200-215 kDa from the FCR-3 strain) released by alkaline borohydride were treated with HNO2 and then analyzed for hexosamines (Table I). More than 90% of the GlcN was sensitive to HNO2, indicating that the carbohydrate moieties of parasite proteins contain nonacetylated GlcN (Table I). Although unlikely, it is possible that the GlcN could have been derived from an N-acetylglucosaminyl moiety by de-N-acetylation during the treatment with alkaline borohydride. Therefore, the carbohydrate moieties of the parasite proteins released by the Pronase digestion were similarly deaminated and then partitioned between water and water-saturated 1-butanol. After deamination, ~90% of the radioactivity remained in the aqueous phase, and the remainder partitioned into the 1-butanol layer. A retreatment of the radioactivity in the organic phase with HNO2 partitioned almost all of the carbohydrate moieties into the aqueous phase. GlcN was barely detectable after deamination of the carbohydrate moieties of parasite proteins. In all parasite proteins analyzed, GlcN was converted into 2,5-AHM (identified as 2,5-anhydromannitol; see below) after treatment with HNO2. These results indicate that the carbohydrate moieties of parasite proteins contain predominantly GlcN. This finding was confirmed for all P. falciparum strains used in this study.
The presence of GlcN in the carbohydrates of parasite proteins was further supported by periodate oxidation studies. Dionex-HPAEC analysis of the acid hydrolysates of periodate-oxidized products revealed that the GlcN residues were almost quantitatively destroyed. However, upon N-acetylation, the GlcN residues were completely resistant to periodate oxidation.
The predominance of GlcN with little or no N-acetylhexosamines in all of the predominantly radiolabeled parasite proteins (from FCR-3, D6, W2, and NF54 strains) suggests that GPI anchors are the major carbohydrate moieties of the parasite proteins. To verify whether this is the case, the parasites were metabolically labeled with [3H]ethanolamine. SDS-PAGE of the cell lysates and fluorography gave a labeling pattern of proteins similar to that observed with [3H]GlcN labeling (data not shown). The Bio-Gel P-4 chromatography of the [3H]ethanolamine-labeled components released from the protein gel bands, either by Pronase or alkaline borohydride, gave elution patterns similar to the carbohydrate moieties obtained from [3H]GlcN-labeled parasite proteins (data not shown).
Analysis of P. falciparum Whole Cell Lysate for Carbohydrate Moieties in ProteinsAlthough N- and O-linked carbohydrates were not detected in the individual parasite proteins separated by SDS-PAGE, it is possible that they are present in very low proportions compared with GPI moieties. Therefore, entire P. falciparum cell lysates were analyzed for these carbohydrate moieties. The [3H]GlcN-labeled parasite lysates were extensively dialyzed to remove all free [3H]GlcN and then precipitated with MeOH. The later step ensures the removal of detergents and any free [3H]GlcN remaining in the lysate. The total parasite proteins were then analyzed for GPI anchors and N- and O-linked carbohydrates by the following three procedures.
Treatment of the whole parasite proteins with alkaline borohydride
quantitatively converted the metabolically labeled radioactivity into
lower molecular weight components. Upon chromatography on Bio-Gel P-4,
these were eluted as broad heterogeneous peaks (Fig. 4A) with ~90% of the radioactivity eluting
at volumes similar to the elution of GPI anchors obtained from the
SDS-polyacrylamide gel bands. The eluted radioactivity was pooled
together as shown in Fig. 4A and analyzed for carbohydrates.
[3H]GlcNAcol or
N-[3H]acetylgalactosaminitol were not detected
in fractions A1, A2, and
A3, by HPAEC analysis, either before or after
acid hydrolysis and re-N-acetylation. However, after acid
hydrolysis, ~20-30% and <5% of radioactivity in fractions
A2 and A3, respectively was recovered as GlcN. The remainder of the radioactivity in fractions A2 and A3 appears to be
noncarbohydrate (see below). Carbohydrate analysis, before and after
treatment with HNO2, indicated that GlcN accounts for
~95% the radioactivity in fraction A1, and
~5% appears to be GlcNAc.
Approximately 6% of the radioactivity was released from the
[3H]GlcN-labeled, delipidated, whole parasite proteins by
digestion with N-glycanase. Upon Bio-Gel P-4 chromatography,
the released material eluted as three distinct peaks (Fig.
4B), B1, B2,
and B3 representing, respectively, 1.2, 2.3, and
2.5% of the total [3H]GlcN incorporated into the
parasite proteins. The eluted radioactivity was pooled as indicated in
Fig. 4B and analyzed for carbohydrates. All three fractions
(B1, B2, and
B3) gave GlcN after acid hydrolysis. However
HPAEC analysis before acid hydrolysis, showed the absence of
hexosamines and N-acetylhexosamines in fraction
B3. Fraction B3 does not
bind to Ag 1-X 12 (H+) resin, suggesting that it is not
free GlcN. However, almost all radioactivity in fraction
B3 was bound to Ag 1-X8 (carbonate) and Ag 4-X4
(base) resins. These results suggest that fraction B3 is a negatively charged (possibly phosphate)
derivative of GlcNAc. Fractions B2 and
B3 gave both GlcN and GlcNAcol on sequential NaBH4 reduction, acid hydrolysis, and
N-acetylation suggesting that they contain reducing end
GlcNAc. Fraction B2 appears to release 1 or 2 residues of Man on treatment with jack bean -mannosidase. On Bio-Gel
P-4, the elution position of fraction B2
corresponds to Man3GlcNAc2. Together, these
results suggest that parasite proteins contain a low level of low
molecular weight N-linked oligosaccharides. Further
characterization of these carbohydrates could not be carried out
because of their low abundance.
Digestion of the [3H]GlcN-labeled parasite proteins with
Pronase and partitioning of the digest between water and 1-butanol gave
~90% of the radioactivity in the organic phase and the remainder in
the aqueous layer. After acid hydrolysis, the materials in both the
organic and aqueous phases gave only [3H]GlcN as the
radioactive sugar. The GlcN residues of the carbohydrates in the
1-butanol phase were almost quantitatively sensitive to HNO2, suggesting that this fraction represents
predominantly GPI anchors. The Bio-Gel P-4 elution profile of the
radioactivity in the aqueous phase is shown in Fig. 4C; the
eluted radioactivity was pooled as shown. Acid hydrolysis of the
fractions C1 and C2, before and after treatment with HNO2, and HPAEC analysis
indicated that both fractions contained only [3H]GlcNAc;
no other radiolabeled sugars were detected. Only 40-50% of the
radioactivity in fraction C2 is accounted for by
[3H]GlcNAc; the remainder appears to be noncarbohydrate,
possibly amino acids or peptides formed from nonspecifically
radiolabeled proteins due to the entry of GlcN into glycolytic pathway
(53). Treatment of Fraction C1 with jack bean
-mannosidase shifted the elution position on Bio-Gel P-4 to a
slightly lower molecular weight region, corresponding to the removal of
two or three Man residues. These results, taken together with those
from alkaline
-elimination and N-glycanase treatment,
suggest that parasite proteins contain a low level of
N-linked oligosaccharides.
To determine whether P. falciparum also contains
unsubstituted terminal residues of GlcNAc, parasites at the trophozoite
and schizont stages were isolated from the Percoll-enriched infected erythrocytes by saponin treatment as described (18). In a separate experiment, [3H]GlcN-labeled parasites were released by
treating the infected erythrocytes with saponin as above. SDS-PAGE and
fluorography showed the presence of radiolabeled proteins in the
parasites but not in the supernatant (data not shown), suggesting that
the parasites were intact. The parasite lysates were galactosylated using UDP-[3H]Gal and bovine milk GalT (50). SDS-PAGE
fluorography (Fig. 5) demonstrated that whereas
ovalbumin and several erythrocyte proteins ranging from 20 to 300 kDa
were galactosylated, none of the proteins with molecular weight >25
kDa were galactosylated in the parasite lysate. However, two distinct
proteins (14.5 and 18 kDa) and a smear at 20-25 kDa were labeled in
the parasite lysate. Galactosylatable proteins with comparable
molecular weights were also present in the lysates of erythrocyte
ghosts, albeit in low proportions (Fig. 5). Considering that the
parasite feeds on erythrocyte components and that many erythrocyte
glycoproteins contain high levels of terminal GlcNAc residues, it is
distinctly possible that the observed labeling is due to erythrocyte
proteins internalized and degraded by the parasite.
P. falciparum was reported to contain a peptide O-glycosidic GlcNAcT that can transfer GlcNAc onto Thr residues of a synthetic peptide, Pro-Tyr-Thr-Val-Val (23). This was investigated by incubating the above synthetic peptide and UDP-[3H]GlcNAc with the parasite lysate as described (23). Product analysis on Bio-Gel P-2 (23), demonstrated that the peptide was not glycosylated to a detectable extent. Several preparations of the parasite lysates and parallel negative controls (without the peptide, and only UDP-[3H]GlcNAc in buffer) were analyzed. In each case, product analysis (23) showed the presence of a radioactive peak corresponding to GlcNAc (at an elution volume higher than the peptide). Approximately the same amount of radioactivity was also eluted from control incubations (uninfected red cell lysates and buffer only), and it appeared to correspond to ~0.5% of free [3H]GlcNAc contaminant present in UDP-[3H]GlcNAc. These results suggest that P. falciparum either does not express peptide O-glycosidic GlcNAcT activity or contains only a very low level of such enzyme activity.
Structural Characterization of Glycans from the GPI Anchors of P. falciparumThe [3H]GlcN-labeled carbohydrate
moieties, isolated by the alkaline borohydride treatment and Bio-Gel
P-4 chromatography, of various parasite protein bands were separately
dephosphorylated with aqueous HF and then deaminated and reduced with
NaBH4. In each case, Man4-AHM was identified as
the major product (>95%) by size exclusion chromatography on Bio-Gel
P-4. The neutral glycans derived from the 200-215-, 82-, and 75-kDa
proteins were each microsequenced by analyzing the products of
exoglycosidase digestion and partial acetolysis using three different
chromatographic techniques (see below). The glycans of the GPI anchors
of 14-, 30-38-, 43-56-kDa proteins were characterized by analyzing on
a Bio-Gel P-4 column before and after treatment with A. saitoi -mannosidase and/or jack bean
-mannosidase
(Table I).
Gel filtration of neutral glycans of individual parasite protein GPI
anchors on calibrated Bio-Gel P-4 columns using Glc as an internal
standard, gave, in all cases, a major peak corresponding to 5.3 GU with
a small amount (<5%) of higher molecular weight components; the
elution positions of the major peaks were similar to the standard
Man4-AHM (data not shown), suggesting that the glycan
moieties consist of four hexoses and one GlcN residue. Digestion of the
neutral glycans with A. saitoi, the 1,2-linkage-specific -mannosidase, shifted the elution position to that of standard Man2-AHM (3.3 GU), indicating the removal of two Man
residues (data not shown). Acetolysis of the neutral glycans under
conditions that preferentially hydrolyzes
1,6-glycosidic bonds, gave
a major and a minor peak (2.4 and 1.7 GU) corresponding to that of
standard Man-AHM and AHM, respectively (not shown). Treatment with jack bean
-mannosidase shifted the elution volume to that of standard AHM
(not shown). Taken together, these results demonstrate that the glycan
moieties of the parasite protein GPI anchors consist of four Man
residues and one GlcN residue.
On Dionex-HPAEC, the neutral glycans of the 200-215- and 82-kDa
protein GPI anchors eluted as single peaks corresponding to the
Man4-AHM standard (data not shown). Treatment of the
neutral glycans with A. saitoi -mannosidase shifted their
retention times to a value comparable to that of standard
Man2-AHM (data not shown). The products of jack bean
-mannosidase coeluted with AHM (not shown).
HPTLC analysis of the GPI anchor glycan cores, isolated from 200-215-
and 82-kDa proteins, showed one major band at an
Rf value identical to Man4-AHM
standard (Fig. 6, lane 4; see also Fig.
7, A and B, lanes 2).
Several slow migrating minor bands representing <5% of the total
radioactivity were also present (see Fig. 7, A and
B, lanes 2). These may represent glycans with unidentified substituents, and they were not further characterized because of their low abundance. Treatment of the glycans with A. saitoi -mannosidase increased the Rf
values to that of authentic Man2-AHM (Fig. 6, lanes
1 and 5). Partial acetolysis gave one major (Man-AHM)
and two minor products (Man2-AHM and AHM) (Fig. 6,
lanes 2 and 6). Digestion with jack bean
-mannosidase gave one major product with the same
Rf value as that of authentic AHM (Fig. 6,
lanes 3 and 7).
The linkage position between the Man residue and the GlcN was determined by periodate oxidation. Compositional analysis using Dionex HPAEC indicated that the GlcN residues of the GPI anchors are quantitatively oxidized by periodate (data not shown). However, upon N-acetylation, the GlcN residues were completely resistant to periodate. These results indicate that GlcN is substituted either at C-4 or at both C-4 and C-6, but not at C-3. However, the stability of this glycosidic bond to partial acetolysis excludes the possibility of a 1,6-linked glycosidic bond between the first Man residue and the GlcN residue. Thus, this linkage position should be 1,4.
The results of the above analyses establish the sequence
Man1-2Man
1-2Man
1-6Man
1-4AHM for the glycan cores of the
parasite protein GPI anchors.
The
carbohydrate moieties of the parasite proteins (200-215 and 82 kDa)
were deaminated, reduced with sodium borohydride, and then subjected to
partial hydrolysis using conditions that do not affect the ethanolamine
phosphate linkage (46, 49). The partial hydrolysates were divided
into two equal parts. In each case, one part was directly
dephosphorylated with aqueous HF and the other part was digested with
jack bean -mannosidase before dephosphorylation. The products
were analyzed by gel filtration on Bio-Gel P-4 (data not shown) and by
HPTLC (Fig. 7).
The partial hydrolysates that were directly dephosphorylated gave five
peaks that correspond to Man4-AHM, Man3-AHM,
Man2-AHM, Man-AHM, and AHM on Bio-Gel P-4 chromatography
(data not shown). HPTLC analysis gave a ladder of Man4-AHM,
Man3-AHM, Man2-AHM, Man-AHM, and AHM (Fig. 7,
A and B, lanes 3). Digestion of the partial hydrolysates with jack bean -mannosidase before treatment with aqueous HF gave a mixture of Man3-AHM and AHM (Fig. 7,
A and B, lanes 4).
Man3-AHM is formed from GPI glycan cores that were
unaffected and those in which the fourth Man was cleaved during the
partial hydrolysis, whereas AHM is formed from glycans in which the
partial hydrolysis cleaved two or more Man residues. These results
demonstrate the presence of a phosphate ester substituent on the third
Man residue from AHM. This substituent is likely to be the conserved
protein-anchoring ethanolamine-phosphate moiety attached to the O-6
position of the third Man residue (for review see Ref. 54).
Jack bean -mannosidase, which removes only the
unsubstituted
-Man residues from the nonreducing end, was used to
identify substituents on the terminal Man residue. The GPI anchors of
200-215- and 82-kDa parasite proteins isolated by mild alkaline
saponification of the Pronase-digested product or those obtained by
treatment with alkaline borohydride were digested with jack bean
-mannosidase before and after treatment with HNO2 and
NaBH4. The products were dephosphorylated with aqueous HF,
and those not already deaminated were then treated with
HNO2 and NaBH4. The GPI anchors that were treated with
-mannosidase after nitrous acid deamination gave Man3-AHM on Bio-Gel P-4 chromatography (data not shown) and
on HPTLC (Fig. 8, lanes 2 and 5).
However, the GPI anchors that were digested with
-mannosidase prior
to nitrous acid deamination and sodium borohydride reduction gave a
mixture of Man4-AHM (50-60%) and Man3-AHM
(40-50%) (data not shown, and Fig. 8, lanes 3 and 6). These results indicate that at least 50-60% of the
nonreducing end Man residues are substituted and that these
substituents were almost quantitatively removed under the conditions of
nitrous acid treatment. The results were reproducible for several
purified preparations of GPI anchors from both 200-215- and 82-kDa
proteins. The GPI anchor samples, those treated and not treated with
HNO2/NaBH4 prior to the
-mannosidase
digestion, were similarly purified on Bio-Gel P-4. Therefore, the
observed results were not due to incomplete removal of the terminal Man
residues by jack bean
-mannosidase, caused by contaminants in
samples that were not treated with HNO2/NaBH4. Thus, the glycan cores of the parasite protein GPI anchors have the
following structure.
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In this study, metabolic labeling with [3H]GlcN established that about 15 proteins of the intraerythrocytic stage P. falciparum are dominantly modified with carbohydrate moieties. Carbohydrate compositional analysis, partitioning of the products of Pronase digestion between water and water-saturated 1-butanol, and structural studies demonstrate that GPI anchors represent the major carbohydrate modification in these parasite proteins.
P. falciparum proteins contain, besides GPI anchor moieties,
low levels of N-glycanase-releasable high Man type and/or
incompletely processed N-linked oligosaccharides and novel
negatively charged GlcNAc residues; the latter was not released by the
reductive alkaline -elimination. These structures together account
for about 6% of the total [3H]GlcN incorporated into the
parasite proteins. Previously, Dieckmann-Shuppert et al.
(23) reported the presence of a small proportion (7-10%) of
N-glycanase-releasable carbohydrates in radiolabeled
parasite proteins. Although the structures were not studied (23), these were likely to be N-linked carbohydrates. Recently, Kimura
et al. (28) reported the existence of N-linked
carbohydrates in the parasites. Considering that GPI anchors represent
the major carbohydrate modification in P. falciparum and
that proteins are modified with GPI anchors on a mol/mol basis, the
level of N-glycosylation in the parasite proteins is very
low. This explains why the presence of N-glycosylation was
evident only on examination of whole cell lysates and was not readily
detectable in individual parasite proteins. The low content of
N-linked carbohydrates in P. falciparum is also
in agreement with the previously reported undetectable levels of
dolichol pyrophosphate-oligosaccharide intermediates, and peptide
N-glycosidic oligosaccharyltransferase activity in the
parasite (27).
The observed low content of N-linked carbohydrates in P. falciparum proteins is not due to the incomplete release by N-glycanase. The amount of radiolabeled carbohydrates released by N-glycanase is comparable with the amounts of glycopeptides recovered in the water phase (<10% of the total), after partitioning of the Pronase digests of the parasite proteins between water and water-saturated 1-butanol. The carbohydrates remaining in the water phase should correspond to N-linked structures, because the GPI anchor moieties are the only other carbohydrates detectable in the parasite proteins (~90%).
In contrast to the results presented here, Kimura et al.
(28) reported that the N-linked carbohydrates represent as
much as 70% of the radiolabeled carbohydrates of ring and early
trophozoite stage P. falciparum, and about 30% in old
trophozoites. These investigators have not considered the presence of
GPI anchor moieties in the parasite proteins. Therefore,
N-linked carbohydrates and other trace amounts of glycans
may have been viewed as the major constituents of the parasite proteins
(28). Despite this difference, our results are in partial agreement
with those of Kimura et al. regarding the structural
features of N-linked carbohydrates (28). In this study,
compositional analysis of the N-glycanase-released carbohydrates, after treatment with NaBH4, gave both GlcN
and glucosaminitol, suggesting that these glycans contain at least two
GlcN residues, one of which is at the reducing end. Based on their size
and susceptibility to jack bean -mannosidase, these oligosaccharides
appear to be Man3-GlcNAc2 and larger high Man type and/or hybrid type structures. Kimura et al. (28) have also found similar structures in addition to
N-glycanase-released chitobiose and single residues of
GlcNAc in the parasite proteins. The latter were not detected in this
study. However, novel, negatively charged residues of GlcNAc apparently
linked to Asn were found in this study.
The observed low content of N-glycosylation in intraerythrocytic P. falciparum is not due to the low abundance of potential N-glycosylation sites in the parasite proteins. The deduced amino acid sequences of erythrocytic stage P. falciparum proteins contain several potential N-glycosylation sites (7, 24, 26). For example, MSP-1 contains as many as 15 potential N-glycosylation sites (7), and the heat shock protein HSP-72 contains four such sites (26). Moreover, a 72-kDa C-terminal peptide of P. falciparum MSP-1, expressed in mammalian cells, contains one to four N-linked oligosaccharide chains per molecule.2 Therefore, it is likely that the low content of N-linked carbohydrates is due to very low N-glycosylation capacity of the parasite. This conclusion is consistent with the previously reported undetectable levels of GDP-Man, dolichol pyrophosphate-oligosaccharide intermediates, and peptide N-glycosidic oligosaccharyltransferase activity (27).
In contrast to the presence of GPI anchors and N-linked carbohydrates, O-linked carbohydrates were not detectable either in the individual protein gel bands or whole P. falciparum lysates. In support of this conclusion, neither free N-acetylhexosaminitols nor oligosaccharides with N-acetylhexosaminitols at the reducing ends were detected after NaOH/NaBH4 treatment of the parasite proteins radiolabeled at different stages of intraerythrocytic development. Galactosylation of lysates of P. falciparum free of red cells using GalT and UDP-[3H]Gal, and SDS-PAGE analysis also did not provide evidence for the abundant presence of O-linked GlcNAc residues in the parasite proteins. In contrast to these results, it was previously reported that O-glycosylation is the major carbohydrate modification in parasite proteins (20-23). The reported carbohydrate structures included single residues of O-GlcNAc (20-23) and oligosaccharides with terminal GlcNAc residues (23). While some of the discrepancies may be due to the lack of consideration given to GPI moieties (20-23, 28), others (28) appear to be due to problems with the technical approaches used (see below).
Previously, Dieckmann-Schuppert et al. (23) used total
proteins from P. falciparum cultures containing 90%
uninfected and 10% parasitized red cells for the release of
O-linked carbohydrates with
NaOH/NaB3H4 and for the galactosylation of
terminal GlcNAc with GalT and UDP-[3H]Gal. These
procedures may have radiolabeled predominantly the carbohydrates of the
native erythrocyte glycoproteins (23) because of the abundance of
O-linked GlcNAc and oligosaccharides bearing terminal GlcNAc
in human erythrocyte glycoproteins (55). This would explain several of
the previously reported contradictory results (23). First, the size and
structural features of the presumed O-linked carbohydrates
of the parasite proteins were strikingly similar to those obtained from
control erythrocyte proteins (23). Second, the 2.5-3 times higher
level of radiolabeling of carbohydrates might have been due to
batch-to-batch variations in radioactivity incorporation into the
carbohydrates of the erythrocytic glycoproteins rather than
radiolabeling of the parasite carbohydrates. Third, the amount of
presumed Gal1-4GlcNAcol, obtained after galactosylation, appeared
to be higher for proteins from control red cells compared with those
from red cells with 10% parasitemia. Fourth, the reported effective
size of Gal
1-4GlcNAcol on Bio-Gel P-4 was 2 GU or less (23), a
figure not in agreement with the reported effective sizes of
galactitol, N-acetylgalactosaminitol, and Hex
1-4GlcNAcol
of 1.5, 2.6, and 3.2, respectively (52), raising doubts about the
authenticity of the characterized structure. Finally, whereas we and
others (18, 28) found that uninfected erythrocytes do not metabolically
incorporate sugars into their proteins to significant levels,
Dieckmann-Shuppert et al. (23) reported that the level of
14GlcN incorporation into proteins of control red cells was
about half the level of incorporation into proteins of red cells with 10% parasitemia (27). The latter is not plausible, considering that
UDP-GlcNAc is not synthesized by uninfected red cells (27).
To avoid high background interference due to the abundance of O-linked carbohydrates in erythrocyte glycoproteins, we performed biochemical analysis with the metabolically radiolabeled P. falciparum proteins separated on SDS-polyacrylamide gels and with lysates of Percoll-enriched parasitized erythrocytes. Galactosylation was carried out with lysates of the isolated parasites. Despite these precautions, interference from red cell glycoproteins was apparent in galactosylation studies (see Fig. 5, lane 2); this is likely due to glycoproteins internalized and degraded by the parasite. The results of metabolic labeling argue against these being parasite proteins, because similar size glycoproteins bearing N- or O-linked carbohydrates were not apparent in the parasite.
It was reported that P. falciparum contains peptide O-glycosidic GlcNAcT activity, based on the use of a synthetic peptide, Pro-Tyr-Thr-Val-Val, as an O-GlcNAc acceptor (23). However, the results of this study demonstrate that neither the lysates of Percoll-enriched parasitized erythrocytes nor the lysates of the parasite isolated by mild saponin lysis of the infected erythrocytes carry out detectable glycosylation of Pro-Tyr-Thr-Val-Val. This discrepancy may be due to very low levels of O-glycosidic GlcNAcT activity in the parasite, because the results of biochemical analysis demonstrate that O-GlcNAc residues are undetectable in the P. falciparum proteins.
Kimura et al. (28) have reported that P. falciparum proteins contain O-glycanase-releasable
carbohydrate(s). Although the structure(s) was not determined, the
released carbohydrate(s) was suggested to be Gal1-3GalNAc (28) in
view of the strict substrate specificity of the enzyme (56, 57). In
contrast to this, the present study demonstrates that GalNAc is not
detectable in the parasite proteins. This result is in agreement with
the previous reports that P. falciparum neither synthesizes
GalNAc nor metabolically utilizes exogenous GalNAc for glycosylation (23) and that the Golgi-resident GalT is undetectable in the parasite
(58). Thus, the occurrence of O-glycanase-releasable carbohydrates in P. falciparum remains to be established.
Although the results of this study indicate that O-linked carbohydrates are undetectable in P. falciparum proteins, O-GlcNAc may be present in the parasite proteins at very low levels, i.e. below the detection limits of the methods used. O-Glycosylation of cytoplasmic and nucleoplasmic proteins with single residues of GlcNAc has been shown to be generally present in almost all eukaryotic cells, and this kind of glycosylation is reported to be a dynamic process, responsive to cellular metabolism (50, 59-61). Therefore, single residues of O-GlcNAc may be present in P. falciparum proteins at very low levels, i.e. <1-2% compared with the total GPI anchors of the parasite proteins, since N-linked carbohydrates representing ~1-2% (<1 chain/~50 molecules of proteins) were distinctly detected. However, single residues of O-GlcNAc are not likely to be present in GPI-anchored cell surface parasite proteins, because plasma membrane proteins, in general, do not undergo cytoplasmic O-glycosylation (61).
In this study, we establish that the GPI glycan cores of multiple
proteins from four different strains of P. falciparum
consist of Man1-2Man
1-2Man
1-6Man
1-4AHM. We also show
that the glycan cores of the GPI anchors contain substituents attached
to the first Man residue from the nonreducing end, and they are
susceptible to the conditions of nitrous acid deamination. The nature
and location of the substituents appear to be unusual and have not been
previously reported in GPI moieties of proteins from other sources.
The GPI anchors of P. falciparum proteins have been reported to be involved in several critical functional roles (28-30, 62). In agreement with these reports, our recent studies suggest that mannosamine, a known inhibitor of GPI biosynthesis (63-65), is lethal to the erythrocytic stage parasite.3
The P. falciparum GPI anchors can induce cytokine release in host macrophages and cause pathological conditions in mice that include transient pyrexia, hypoglycemia, and lethal cachexia (32). Recently, it has been reported that both the GPI anchors MSP-1 and MSP-2 induce nitric-oxide synthase expression in macrophages and vascular endothelial cells by a protein-tyrosine kinase-dependent and protein kinase C-dependent signaling pathway (36). The GPI anchors of these proteins are also reported to up-regulate the levels of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin expression in vascular endothelial cells as well as to cause increased leukocyte and parasite cytoadherence to vascular endothelial cells via tyrosine kinase-dependent signal transduction (35). The structural basis for this broad bioactivity of the GPI anchors of P. falciparum proteins is not known. However, it has been suggested that the phosphatidylinositol moiety of the parasite GPI alone is not sufficient for protein-tyrosine kinase-induced cell signaling and that the glycan moiety is also involved in this activity (36).
We thank Armida Torres-Duarte for cell cultures and metabolic labeling, Dr. Alvaro A. Serrano (University of Sao Paulo, Brazil) for Man4-AHM and Man-AHM ladder, and Dr. V. P. Bhavanandan (Hershey Medical Center, Hershey, PA) for 3H-labeled Glc oligomers.