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Originally published In Press as doi:10.1074/jbc.M800725200 on April 16, 2008

J. Biol. Chem., Vol. 283, Issue 26, 18355-18364, June 27, 2008
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Unique Asn-linked Oligosaccharides of the Human Pathogen Entamoeba histolytica*Formula

Paula Magnelli{ddagger}, John F. Cipollo§, Daniel M. Ratner{ddagger}, Jike Cui{ddagger}, Daniel Kelleher, Reid Gilmore, Catherine E. Costello§, Phillips W. Robbins{ddagger}, and John Samuelson{ddagger}1

From the {ddagger}Department of Molecular and Cell Biology and the §Mass Spectrometry Resource, Department of Biochemistry, Boston University Medical Center, Boston, Massachusetts 02118-2526 and the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605-2324

Received for publication, January 28, 2008 , and in revised form, March 21, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man5GlcNAc2), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man5GlcNAc2, which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc1Man5GlcNAc2, which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive {alpha}-mannosidase trims some N-glycans to biantennary Man3GlcNAc2. Complex N-glycans of E. histolytica are made by the addition of {alpha}1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man5GlcNAc2, which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Entamoeba histolytica is the protist (single cell eukaryote) that causes millions of cases of amebic dysentery and liver abscess in regions where its fecal-oral spread cannot be prevented (1, 2). Asn-linked glycans (N-glycans) of E. histolytica are of great interest for seven reasons.

First, E. histolytica is missing many of the glycosyltransferases that make lipid-linked precursors to N-glycans and so makes a Man5GlcNAc2-PP-dolichol rather than Glc3Man9GlcNAc2-PP-dolichol, which is present in most animals, plants, and fungi (3, 4). Second, E. histolytica N-glycans contribute to the quality control of protein folding in the endoplasmic reticulum (ER)2 (4-6). In particular, E. histolytica has UDP-Glc:glycoprotein glucosyltransferase, calreticulin, glucosidase II, and ERGIC-53, which are the essential components of an N-glycan-dependent quality control system (4-6).

Third, the E. histolytica oligosaccharyltransferase (OST), which transfers N-glycans from the dolichol pyrophosphate-linked precursor to Asn on the nascent peptide in the lumen of the ER is composed of four subunits rather than seven or eight subunits found in metazoan, fungi, and plants (7). As a result, the E. histolytica OST has different kinetics than the OSTs of higher eukaryotes (8). As well, the E. histolytica OST prefers to transfer N-glycans, which resemble its own (Man5GlcNAc2), rather than the longer N-glycans of metazoa and fungi (Glc3Man9GlcNAc2) (8).

Fourth, E. histolytica has a limited set of nucleotide sugar transporters (UDP-Gal and UDP-Glc), which transport activated sugars into the lumen of the ER and Golgi (9). UDP-Gal and UDP-Glc are used to make unique O-phosphodiester-linked glycans of E. histolytica proteophosphoglycans (10) and may be used to make complex N-glycans.

Fifth, E. histolytica causes disease when the protist uses a Gal- and GalNAc-binding lectin on its surface to adhere to mucins on the surface of host colonic epithelial cells and then lyses host cells by means of secreted proteases and pore-forming peptides (1, 2, 11, 12). Heavy subunits of the E. histolytica Gal/GalNAc lectins have 7-14 potential sites for N-linked glycosylation, and inhibition of N-glycan synthesis results in a Gal/GalNAc lectin that is unable to bind its target (11).

Sixth, the plant lectin concanavalin A binds to glycoproteins on the surface of E. histolytica and aggregates them into caps that are shed into the medium (13). This result suggests the possibility that E. histolytica may have on its surface high mannose N-glycans. In a similar way, gp120 of HIV has on its surface N-glycans composed of Man9GlcNAc2 (14). These high mannose N-glycans on gp120 are the target of an anti-HIV human monoclonal antibody (2G12) and of bacterial anti-retroviral lectins (cyanovirin-N and scytovirin) (15, 16).

Seventh, although N-linked glycans of Giardia lamblia and kinetoplastids (e.g. Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana) have been characterized, N-glycans of the vast majority of protists (e.g. E. histolytica, Trichomonas vaginalis, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum) remain uncharacterized (3, 17-19). Characterization of E. histolytica N-glycans then may lead to a better understanding of the diversity of N-linked glycosylation in protists and may identify novel sugar linkages that have not previously been identified in higher eukaryotes.


Figure 1
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  		FIGURE 1.
E. histolytica trophozoites make numerous N-glycans from the truncated Man5GlcNAc2 precursor transferred by the OST (3, 8). The most abundant N-glycan in each size range is listed first (e.g. H3.1, H4.1, etc.). Structures were identified by multiple techniques including in vivo and in vitro labeling, size separation on Bio-Gel P-4 and HPAEC, glycosylhydrolase digestions, monosaccharide analysis, and mass spectrometry. An asterisk marks the linkage between Glc and Gal, which could not be determined experimentally.

 
Here, biochemical methods including in vivo and in vitro labeling, as well as mass spectrometry, were used to characterize E. histolytica N-glycans. E. histolytica N-glycans include unprocessed Man5GlcNAc2, which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bioinformatic Predictions—The predicted proteins of the E. histolytica genome, which has been extensively sequenced, were searched with BLASTP and representatives (e.g. Saccharomyces, Homo, or Escherichia) of each of 79 glycosyltransferase families and 104 glycosylhydrolase families present in the data base of carbohydrate-active enzymes (CAZy) (20-22). Putative E. histolytica glycosyltransferases and glycosylhydrolases were compared with proteins in the NR Database and to the conserved domain data base at the NCBI using PSI-BLAST. Signal peptides and transmembrane helices of E. histolytica proteins were predicted using SignalP and TMHMM, respectively (23, 24).

Reagents—[2-3H]Man (30 Ci/mmol), [U-14C]Glc (200 mCi/mmol), UDP-[3H]Gal (5.8 Ci/mmol), and UDP-[3H]Glc (300 mCi/mmol) were from American Radiolabeled Chemicals (St. Louis, MO). Peptide:N-glycanase F (PNGase F) was from New England Biolabs. Jack bean {alpha}-mannosidase and almond β-glucosidase were from Sigma. Coffee bean {alpha}-galactosidase, β1,3,4,6-galactosidase, and β-glycosidase II (β-galactosidase and β-glucosidase activities) were from Calbiochem. Aspergillus saitoi {alpha}1,2-mannosidase, bovine testes β-galactosidase, Jack bean hexosaminidase, Man3GlcNAc2, and Man1GlcNAc2 standards were from Glyko (San Leandro, CA). Maltase and amyloglucosidase ({alpha}-glucosidases) were from Roche Applied Science. Golgi endomannosidase (25) was a generous gift from Dr. Robert Spiro (Harvard Medical School). Cyanovirin-N and scytovirin were generous gifts of Barry O'Keefe (NCI-Frederick, National Institutes of Health).


Figure 2
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  		FIGURE 2.
The complexity of N-glycans made by E. histolytica trophozoites increases dramatically with time. E. histolytica trophozoites were incubated with [2-3H]Man for 10, 20, 60, and 150 min, and N-glycans were released with PNGase F and separated by Bio-Gel P-4 filtration (A-D), where Hexn indicates their size. Isomers with the same number of hexoses were further isolated on an HPAEC-PA100 column (E-H), with the retention time (ret.; min) shown in italic and the identifier name from Fig. 1 (e.g. H5.1) underlined below. After 10-min labeling (A and E), the predominant E. histolytica N-glycans were unmodified Man5GlcNAc2 and the glucosylated product of UDP-Glc:glycoprotein glucosyltransferase (Glc1Man5GlcNAc2) (H6.1). As shown in the inset in A, Glc1Man5GlcNAc2 is by far the most abundant N-glycan in the presence of the glucosidase II inhibitor castanospermine (CTS). After 20-min labeling (B and F), two mannosidase digestion products are apparent: Man4GlcNAc2 (H4.1) and Man3GlcNAc2 (H3.1). After 60-min labeling (C), complex N-glycans are apparent in Hex6 and Hex7 pools. After 150-min labeling (D and G), novel, complex N-glycans are present in all of the pools (see further characterization in Fig. 4). frct., fraction.

 
In Vivo Labeling Conditions—E. histolytica strain HM1:IMSS was grown axenically in TYI-S-33 medium containing 0.1% (w/v) Glc to minimize the storage of glycogen, in which fragments otherwise contaminate N-glycan extracts (26). E. histolytica cultures from five 32 cm2 flasks were combined, centrifuged, and transferred to a microcentrifuge tube with 0.5 ml of radiolabeling medium (the same as described above, excluding Glc and containing 200 µCi of [2-3H]Man or 2 mCi of [14C]Glc) (3). Amebas were incubated for the indicated time at 37 °C and then washed in PBS several times before processing.

Extraction of N-Glycans—Non-incorporated radiolabel was removed by washing the cell pellet with 50% methanol. Cells were lysed with a Dounce homogenizer in 4 ml of Tris-HCl buffer, pH 8. To reprecipitate proteins, the pH was adjusted to 5 with acetic acid, and 4 ml of methanol was added. Tubes were chilled at -20 °C for 4 h and centrifuged at 14,000 rpm for 10 min. This step was repeated twice to wash out soluble glycans such as glycogen fragments. The methanol-denaturated protein pellet was dispersed and treated with 5,000 units of PNGase F in acetate buffer, pH 5, at 37 °C for 16 h. The suspension containing the released N-glycans in solution was adjusted to 50% methanol to precipitate large carbohydrates and proteins. After 4 h at -20 °C, the supernatant was cleared by centrifugation and dried. Oligosaccharides were further purified on a porous graphitic carbon Hypercarb column (Thermo Keystone) and washed with 3 ml of distilled water. The N-glycans, which were eluted with 30% acetonitrile, 0.1% trifluoroacetic acid, were dried and resuspended in 300 µl of water.

In Vitro Labeling of N-Glycans—Intact E. histolytica vesicles from freshly harvested cultures were prepared as described for nucleotide sugar transport assays (9), excluding separation ER enriched vesicles from light Golgi enriched vesicles. Labeling of endogenous acceptors was performed by incubating the E. histolytica vesicles with UDP-[3H]Gal and UDP-[14C]Glc as described (9). The vesicle preparation still carried a significant amount of cytosolic epimerase activity, which converts UDP-Gal to UDP-Glc, in a reversible reaction. Because of this activity, [3H]Glc was incorporated into E. histolytica glycoproteins when labeling with UDP-[3H]Gal and vice-versa. Therefore, specific labeling was not possible. The labeling of glycans was started by mixing 500 µl of vesicle suspension and 500 µl of reaction buffer (10 mM MnCl2, 10 mM MgCl2, 10 mM CaCl2, 0.5 M sucrose, 30 mM triethanolamine, pH 7.2, 5 µM UDP-Gal, and 5 µM UDP-Glc and 30 µCi of each radiolabeled precursor). After 45 min at 37 °C, the reaction was diluted with 1 volume of 100 µM UDP-Gal and UDP-Glc and separated into 100-µl aliquots, each of which was precipitated with 900 µl of 4% perchloric acid. After chilling on ice for 30 min, the preparation was centrifuged at 16,000 x g for 30 min, and the pellets were combined and neutralized. The pellets were resuspended with 2 ml of 10 mM Tris-HCl buffer, pH 8, in 50% methanol and homogenized in a 3-ml Dounce. After 2 h at -4 °C, the suspension was centrifuged at 14,000 x g for 10 min, and the pellet was washed twice with Tris-HCl buffer, pH 8, to remove traces of methanol prior to release of N-glycans by digestion with PNGase F (see conditions above).


Figure 3
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  		FIGURE 3.
The abundance of Glc in complex N-glycans of E. histolytica trophozoites increases with their length. A, E. histolytica N-glycans labeled with [U-14C]Glc and separated by Bio-Gel P-4 filtration. The number of hexoses in each glycomer is indicated (Hexn), according to calibrated standards prepared from Man5GlcNAc2 digested with jack bean {alpha}-mannosidase and A. saitoi {alpha}1,2-mannosidase. B, relative abundance (weight percentage) of neutral sugars for each peak after acid hydrolysis, as determined by HPAEC chromatography. fract., fraction.

 
Sample Preparation for Mass Spectrometry—The starting material to isolate N-glycans for mass spectrometry was a cell pellet obtained from 60 flasks of E. histolytica culture. First, lipids were extracted three times with 30 ml of chloroform: methanol (1:1) and three times with 30 ml of chloroform:methanol:water (10:10:3). Soluble carbohydrates (mostly glycogen fragments) were removed by extracting three times with 20 ml of 10 mM Tris-HCl buffer, pH 8, and reprecipitating with 1 volume of chilled methanol for 4 h at -20 °C. After a final wash in buffer, the pellet was digested with 1 µg of TPCK-treated trypsin in 1 ml of 10 mM Tris-HCl buffer, pH 8, for 16 h at 37 °C. After boiling to inactivate the protease, the suspension was digested with 20 x 103 units of PNGase F in the presence of phenylmethylsulfonyl fluoride for 16 h at 37 °C. The mixture was precipitated with 50% methanol, and the supernatant was purified through a porous graphite column, followed by passage through a 3-ml Amberlite mixed bed ion exchange column (H+/acetate form). Finally, the N-glycan extract was dried and resuspended in 300 µl of water.

Chromatography—A Bio-Gel P-4 superfine mesh column of 1 x 120 cm was equilibrated in 0.1 M acetic acid, 1% n-butyl alcohol. 300 µl of glycan sample was applied and run at a constant flow rate of 5 ml/h, and 1.3-ml fractions were collected. Radioactivity was determined by liquid scintillation counting. The distribution coefficient (Kd = (Ve - Vo)/(Vt - Vo)) was determined using the glycan elution volume (Ve) relative to the elution of cytochrome C (marker for the exclusion volume; Vo) and glucose (marker for the total volume; Vt). GlcNAc, diacetylchitobiose, and radiolabeled Man5GlcNAc2 (prepared from the dolichol-linked precursors of {Delta}alg3 Saccharomyces cerevisiae (4)) were used to calibrate the system. For non-radiolabeled samples, peaks were collected according to the Kd of equivalent radiolabeled specimens.

To separate isomers with the same molecular mass, fractions obtained by gel filtration were separated by high performance anion exchange chromatography (HPAEC) with a pulse amperometric detector in a Dionex LC20 instrument through a PA100 column (250 x 4 mm) equilibrated in 150 mM NaOH. The flow rate was 0.6 ml/min with a sodium acetate gradient from 12.5 mM (0-3 min) to 50 mM (at 31 min) and finally to 175 mM (at 70 min). A desalter membrane was installed to remove sodium ions, neutralizing the carbohydrate-containing eluent. Fractions of 0.3 ml were collected, and aliquots were taken for scintillation counting. The retention time of each isomer was determined. The system was calibrated with known N-glycans (Man3GlcNAc2, Man1GlcNAc2, and Man5GlcNAc2 prepared from {Delta}alg3 S. cerevisiae cells) and with the series laminaribiose, laminaritriose, and laminaritetraose (routinely used as internal markers). Non-radiolabeled samples for mass spectrometry were isolated on a 250 x 9 mm semi-preparative PA100 column (flow rate of 1.5 ml/min, collecting 0.7-ml fractions) calibrated and operated (excluding the internal markers) as described above. Carbohydrate-containing peaks were detected by a pulse amperometric detector, and the corresponding fractions were pooled and dried.

Ion Exchange Mini-columns—The resins used were Dowex 50 (H+ form), Dowex 1 (formate form), and Amberlite mixed bed (IRA-400 H+ form and IR-120 acetate form). Columns (0.5 x 2.5 cm) were equilibrated in deionized water. The glycan sample (0.5 ml) was applied followed by water washes, and fractions of 0.25 ml were collected for scintillation counting. The retention behavior of glycans was compared with neutral or charged standards ([2-3H]Man, [3H]GlcN, [3H]Glc 1-phosphate, and UDP-[3H]Glc).

Glycosylhydrolase Digestions—All the glycosylhydrolase digestions were performed in a volume of 100 µl for 16 h at 37 °C. The conditions were adjusted by digestion of known glycans to assure a complete reaction. A final concentration of 100 mM potassium phosphate buffer, pH 6.5, was used for coffee bean {alpha}-galactosidase, maltase, and amyloglucosidase; 100 mM sodium acetate buffer, pH 4.8, was used for Jack bean {alpha}-mannosidase, β-glucosidase, β1,3,4,6-galactosidase, {alpha}1,2-mannosidase, β-glycosidase II, bovine testes β-galactosidase, and Jack bean hexosaminidase digestions. A Golgi endomannosidase reaction was performed in 100 mM Na-MES buffer, pH 6.5, supplemented with 0.1% Triton X-100 and 50 µg/ml bovine serum albumin.


Figure 4
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  		FIGURE 4.
Characterization of the E. histolytica N-glycans by in vivo labeling and glycosylhydrolase digestions. E. histolytica N-glycans, which were prepared as in Fig. 2, were treated with glycosylhydrolases, and digestion products were identified according to retention times (r.t.) of known standards (e.g. Man3GlcNAc2). For isomers H6.2, H6.5, and H7.1, the dashed line enclosing the Man{alpha}1,6-arm indicates what corresponds to the residual fragment 11.5, which was fully characterized in a separate [U-14C]Glc-labeling experiment. Note that jack bean {alpha}-mannosidase (JBAM) removes an exposed Man{alpha}1,3- (as in isomer H4.3), but it does not remove the Man{alpha}1,6-unless the Man{alpha}1,3-arm is digested first. Hence, isomer H4.2 becomes susceptible to jack bean{alpha}-mannosidase digestion only after removal of Gal{alpha}1,2- by {alpha}-galactosidase. The mode of action of jack bean {alpha}-mannosidase (37) supports our assumption that the different sensitivities of H4.2 and H4.3 are due to accessibility of {alpha}-linked Man residues. In accordance with this, the underlying structures of other isomers were inferred as well. ASAM, A. saitoi {alpha}1,2-mannosidase.

 
Monosaccharide Composition—Radiolabeled samples were dried, resuspended in 300 µl of 2 N HCl in microcentrifuge tubes flushed with N2 to minimize oxidation, and hydrolyzed for 2 h at 90 °C. Acid was removed by several rounds of evaporation under N2. The neutral sugars were separated by HPAEC on a MA1 250 x 4 mm column (flow rate of 0.4 ml/min, collecting 0.2-ml fractions) with an NaOH gradient from 100 mM (0-3 min) to 850 mM (to 45 min). GlcN, Glc, Man, and Gal were used as internal standards.

Mass Spectrometry—Oligosaccharides were permethylated as described previously (27). MALDI-TOF MS was performed on a Bruker Reflex IV mass spectrometer in positive reflectron mode. Between 20 and 50 pmol of sample dissolved in 20% acetonitrile was applied to the MALDI target with an equal volume of 2,5-dihydroxybenzoic acid (20 mg/ml) in 20% acetonitrile with 10 mM sodium acetate added as a cation source. The spectra resulting from 150 and 200 shots from a 337-nm nitrogen laser were summed. The laser pulse was 3 ns. Collision-induced dissociation fragmentation data were collected using a QStar Pulsar i quadruple orthogonal time-of-flight mass spectrometer (Applied Biosystems Inc., Framingham, MA) equipped with an electrospray ionization source. Capillaries were pulled to a 1-micron orifice diameter. Argon (3 p.s.i.) was used as the collision gas for MS/MS experiments. The range of operator-controlled collision voltages was 35-90 V. Between 2 and 3 pmol of sample were consumed during each MS/MS experiment. The mass spectrometer parameters were as follows for MS/MS experiments: DP1, 75.0 V; FP, 245.0 V; DP2, 30.0 V; CG, 3.0 p.s.i.; IRD, 6.0 V; IRW, 5.0 V; GS1, 5.0 p.s.i.; GS2, 10.0 p.s.i.; and CUR, 12.0 p.s.i. The ion spray voltage was between 4,000 and 4,500 V. For pseudo MS/MS/MS experiments, the following parameters were used: DP1, 130.0 V; FP, 300.0 V; DP2, 40.0 V; CG, 3.0 p.s.i.; IRD, 6.0 V; IRW, 5.0 V; GS1, 5.0 p.s.i.; GS2, 10.0 p.s.i.; and CUR, 12.0 V. Nomenclature is that of Domon and Costello (28) unless otherwise indicated.

In Vitro OST Assay—Total cellular membranes of E. histolytica were incubated for 2-90 min at 37 °C with the membrane-permeable tripeptide acceptor 5 µM N{alpha}-acetyl-N-125I-Tyr-Thr-NH2 (NYT) in the presence of deoxynojirimycin to ensure that the glycopeptide products were not degraded by glucosidases I and II (29, 30). In some experiments, swainsonine was added to determine the effect of endogenous E. histolytica mannosidases on glycopeptides. Glycopeptide products were collected by binding to immobilized concanavalin A and separated by HPLC using an aminopropyl silica column. Glycopeptide standards (Man5GlcNAc2-NYT and Man9GlcNAc2-NYT) were prepared using the Saccharomyces OST and purified dolichol-linked oligosaccharides (31).

Cyanovirin-N Labeling and Capping of E. histolytica—Freshly harvested E. histolytica were washed three times with PBS and incubated with 20 µg/ml BODIPY-labeled cyanovirin-N for 30 min at 4 °C (14, 15). Unbound cyanovirin-N was removed by rinsing twice with PBS. For surface labeling, E. histolytica were immediately fixed with 2% paraformaldehyde in PBS for 10 min at 4 °C. To determine whether cyanovirin-N aggregates and forms caps on the surface of E. histolytica, we incubated cyanovirin-N-bound cells in PBS for 15 min at 37 °C and subsequently fixed them in 2% paraformaldehyde. For labeling the internal membranes and surface of E. histolytica, amebas were fixed in 2% paraformaldehyde and 0.1% Triton X-100. Organisms were visualized with a DeltaVision deconvoluting microscope (Applied Precision, Issaquah, WA) with the help of Landon Moore of the Department of Genetics and Genomics at Boston University School of Medicine. Images were taken at x100 primary magnification and deconvolved using Applied Precision softWoRx software.


Figure 5
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  		FIGURE 5.
The anti-retroviral lectin cyanovirin-N, which recognizes high Man N-glycans (15, 16), binds to the surface E. histolytica trophozoites and forms caps. A, cyanovirin-N binds to terminal {alpha}1,2-linked Man, which is present on each of the three arms of Man9GlcNAc2 and is also present on the single arm of Man5GlcNAc2. B, cyanovirin-N binds to the surface and to vesicular membranes of fixed and permeabilized E. histolytica. n, nucleus. C, cyanovirin-N is evenly distributed on the surface of a live E. histolytica incubated with the lectin in the cold, so it cannot cap. D, cyanovirin-N is capped on the surface of a live E. histolytica that is allowed to warm up to 37 °C for 10 min. The negative control was scytovirin (a lectin that binds to an intact D3 arm, which is absent in E. histolytica N-glycans). E. histolytica incubated with scytovirin were not labeled (data not shown).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Brief Summary of the Experimental Strategy—The structures of E. histolytica N-glycans, which are shown in Fig. 1, were determined by in vivo labeling with [2-3H]Man (shown in Fig. 2) or [U-14C]Glc (shown in Fig. 3) or by the separation of N-glycans released by PNGase F on Bio-Gel P-4 and HPAEC, followed by digestion with specific glycosylhydrolases (shown in Fig. 4). Alternatively, E. histolytica N-glycans were identified by in vitro labeling of E. histolytica membranes with nucleotide sugars (nucleotide sugar transfer assays shown in Fig. 6) or with a radiolabeled tripeptide (OST assays shown in Fig. 7). In addition, E. histolytica N-glycans were examined by mass spectrometry (shown in Figs. 8 and 9). These strategies allowed us to identify each E. histolytica N-glycan by multiple methods. Finally, we localized unprocessed N-glycans on the surface of E. histolytica using the anti-retroviral lectin cyanovirin-N (shown in Fig. 5).

The Most Abundant N-Glycan of E. histolytica Is Unprocessed Biosynthetic Man5GlcNAc2—The most abundant N-glycan of E. histolytica after in vivo labeling for 20-150 min with [3H]Man or for 3 h with [14C]Glc (Figs. 2 and 3) was unprocessed, biosynthetic Man5GlcNAc2 (H5.1 in Fig. 1), which was characterized by mannosidase digestions (Fig. 4). When E. histolytica were pulse-labeled for 10 min with [2-3H]Man, washed, and chased for 3 h with unlabeled Man, biosynthetic Man5GlcNAc2 was still the most abundant N-glycan (data not shown). This result shows that many E. histolytica N-glycoproteins are not modified by Golgi-type glycosylhydrolases and glycosyltransferases.

Man5GlcNAc2 on the Surface of E. histolytica Trophozoites Is Bound and Capped by the Anti-retroviral Lectin Cyanovirin-N—The presence of biosynthetic Man5GlcNAc2 (H5.1) on the surface of cultured E. histolytica was demonstrated using the anti-retroviral lectin cyanovirin-N (Fig. 5) (15). Cyanovirin-N is specific for the Man{alpha}1,2-Manon the single D1 arm of Man5GlcNAc2 (E. histolytica N-glycans) or on three arms of Man9GlcNAc2 (N-glycans of gp120) (Fig. 5A). Cyanovirin-N was bound to the surface and vesicular membranes of fixed and permeabilized E. histolytica (Fig. 5B). Cyanovirin-N was also bound evenly to glycoproteins on the surface of chilled but living E. histolytica (Fig. 5C). When E. histolytica were warmed to 37 °C, cyanovirin-N aggregated into caps (Fig. 5D), which resemble those formed by the lectin concanavalin A on the E. histolytica surface (11, 13). As a negative control, scytovirin, another anti-retroviral lectin that is specific for Man on the upper (D3) arm of Man9GlcNAc2, which is absent from E. histolytica N-glycans, did not bind to the surface or vesicles of E. histolytica (data not shown) (15).

GlcMan5GlcNAc2, the Product of UDP-Glc:Glycoprotein Glucosyltransferase Involved in N-Glycan-dependent Quality Control of Protein Folding, Is Also Present in Mature N-Glycans of E. histolytica—At the earliest time points of metabolic labeling with [2-3H]Man (Fig. 2A), E. histolytica N-glycans were composed of Man5GlcNAc2 (H5.1) and Glc1Man5GlcNAc2 (H6.1). Glc1Man5GlcNAc2 was markedly increased, whereas Man5GlcNAc2 was decreased, when E. histolytica trophozoites were labeled in the presence of castanospermine, which is a glucosidase II inhibitor (inset in Fig. 2A) (30). These results are consistent with the presence of all the components of N-glycan-dependent quality control of protein folding in E. histolytica (4-6). See supplemental Table 1 for a list of the E. histolytica glycosylation-related genes.

When E. histolytica membranes were incubated in vitro with UDP-[3H]Glc, the most abundant N-glycan was also Glc1Man5GlcNAc2 (Fig. 6, B and E). Glc1Man5GlcNAc2 was also present in relatively high amounts in mature glycoproteins, indicating that this N-glycan is not just a transient species in E. histolytica (Fig. 2, D and H).

Some E. histolytica N-Glycans Are Trimmed Back to Man3GlcNAc2 and Man4GlcNAc2 by a Swainsonine-sensitive Mannosidase—The activity of the E. histolytica {alpha}-mannosidase was inferred by the accumulation of Man3GlcNAc2 (H3.1) and Man4GlcNAc2 (H4.1) after labeling E. histolytica in vivo for longer times (20-150 min) with [3H]Man (Fig. 2). Man4GlcNAc2-NYT and Man3GlcNAc2-NYT were also present after 2-30 min when intact E. histolytica membranes were incubated with the OST substrate NYT (Fig. 7) (10, 29, 31). The E. histolytica mannosidase activity was inhibited by swainsonine, which has been used previously to inhibit class II mannosidases (Fig. 7) (30). In contrast, the E. histolytica mannosidase was not inhibited by deoxymannojirimycin, which inhibits class I mannosidases that are absent from E. histolytica (data not shown).


Figure 6
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  		FIGURE 6.
Synthesis of complex N-glycans in vitro by incubating E. histolytica membranes with UDP-[3H]Glc and UDP[3H]Gal. A, a Bio-Gel P-4 filtration chromatogram of N-glycans is shown. B, isomer separation by HPAEC in PA100 is shown. ret., retention. C, identification of the transferred monosaccharide after total hydrolysis by HPAEC in MA1 is shown. The presence of Gal in the Hex4 isomers and in Hex5 eluting at 17 min is consistent with the {alpha}-galactosidase digests in F. In D-F, untreated glycans are marked with closed circles, and the products of glycosylhydrolases are marked with open circles. D, one Gal1Man3GlcNAC2 isomer (H4.3, at 10.5 min) is digested with jack bean {alpha}-mannosidase (JBAM), whereas the other isomer (H4.2, at 11.5 min) is not. E, Glc1Man5GlcNAC2 (H6.1, at 22 min) is digested to a disaccharide (asterisk) by endomannosidase. F, both Gal1Man3GlcNAC2 isomers (H4.2. and H4.3 at 10.5 and 11.5 min), as well as Gal1Man4GlcNAC2 (H5.2 at 17 min), digest with {alpha}-galactosidase. In contrast, Glc1Gal1Man3GlcNAC2 (H5.6 at 19 min) is not digested by {alpha}-galactosidase. G, the table summarizes these results. Isomer H5.2 was fully characterized using complementary data acquired in a separate [23H]Man-labeling experiment (Fig. 2). frct., fraction; Endomann, endomannosidase; comp, composition; ident, identity.

 
The largest oligosaccharide attached to the radiolabeled tripeptide (NYT) in vitro in the presence of UDP-Gal, UDP-Glc, and swainsonine is Glc1Man5GlcNAc2 (H6.1) (6). These results suggest that the OST, the mannosidase, and the UDP-Glc:glycoprotein glucosyltransferase are all present in the ER, whereas the Gal- and Glc-transferases (see below) are in a distinct organelle (likely the Golgi apparatus).

Elongation of E. histolytica Complex N-Glycans Is Initiated by Addition of {alpha}-Linked Gal to Either Arm of Man3-5GlcNAc2—E. histolytica membranes incubated with UDP-[3H]Gal or UDP-[3H]Glc, the two nucleotide sugars that are transported by E. histolytica membranes (9), produced abundant products ranging from Hex4GlcNAc2 (Hex4) to Hex7 (Fig. 6). In contrast, radiolabeled products were not formed when E. histolytica membranes were incubated with radiolabeled GDP-Man or GDP-Fuc (data not shown). Indeed, monosaccharide analysis of N-glycans of E. histolytica labeled in vivo with [14C]Glc revealed Man, Gal, and Glc but did not show other hexoses or deoxyhexoses (Fig. 3B). Treatment with hexosaminidase, along with mass spectrometry results, showed that N-glycan extensions do not contain hexosamines. All of the N-glycans of E. histolytica were neutral oligosaccharides (data not shown) lacking charged sugars (e.g. sialic acids) or other modifications such as sulfation or phosphorylation.


Figure 7
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  		FIGURE 7.
E. histolytica membranes contain a swainsonine-sensitive {alpha}-mannosidase. Glycopeptides produced by incubating intact E. histolytica membranes with a radiolabeled tripeptide acceptor (NYT) were captured on concanavalin A and resolved by HPLC. HPLC profiles show glycopeptides from assays incubated in the presence (A) or absence (B and C) of swainsonine for 2 min (B) or 30 m in (A and C). Standards include Man5GlcNAc2-NYT, Man9GlcNAc2-NYT, and a glycopeptide mix, Man3-9GlcNAc2-NYT.

 


Figure 8
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  		FIGURE 8.
MS2 collision-induced dissociation spectrum of the Hex3HexNAc2 isolated at m/z 597. 33, [M+2Na]2+. A, the diagram shows the origin of fragments from the two isomers present in this pool (see Fig. 2G). A secondary fragment, Y3{alpha}/Y3β/B3 at m/z 458.21 (B), is consistent with archetypal biantennary Man3GlcNAc2. Cross-ring fragments 0,4A2, m/z 301.14 and 3,5A2, m/z 329.16 (C) define the Man{alpha}1,6-arm. A low abundance 1,3A2 fragment at m/z 315.16 (C) indicates the presence of an isomer bearing a terminal 1,2-linked Gal (H3.2). A Y3/0,2A4 fragment ion seen at m/z 375.17 (B) is consistent with the core region of a linear isomer. No signal is seen at m/z 505.2 or 533.2 (for a substituted Man{alpha}1,6-). Therefore, the structure for H3.2 indicates a missing Man{alpha}1,6-arm, with the remaining Man{alpha}1,3-arm extended with Gal{alpha}1,2-. These results are consistent with the glycosylhydrolase digestion data (Fig. 4).

 
As shown by sensitivity to {alpha}-galactosidase but not to β-galactosidase, a single {alpha}-linked Gal was added to the terminal Man on either or both arms of Man2-5GlcNAc2 when E. histolytica were labeled in vivo or in vitro (e.g. H3.2, H4.2, H4.3, H5.2, H5.4, and H6.4) (Figs. 3 and 4). Fig. 8B shows the signature of a terminal 1,2-linked hexose (a 1,3A2 fragment that cannot originate from Man3GlcNAc2) (H3.1). This result indicates that the minor Hex3 isomer (H3.2) is indeed elongated with an {alpha}1,2-linked Gal. The spectrum for Hex6 N-glycans (Fig. 9) also shows the cross-ring 1,3A2 fragment, indicating the same Gal configuration (e.g. H6.3 to H6.5).

In Many Complex N-Glycans of E. histolytica, Gal Is Capped by One or More Glc Residues—The evidence for complex E. histolytica N-glycans containing Gal and Glc included the following. With increasing time, Hex7 to Hex10 were present in N-glycans of E. histolytica labeled in vivo with [2-3H]Man (Fig. 2) or [14C]Glc (Fig. 3). As the size of N-glycans increased, the percentage of Glc in the oligosaccharides increased (Fig. 3B), and these larger N-glycans (e.g. H5.3, H6.2, H6.5, and H7.1) were resistant to {alpha}- and β-galactosidases, consistent with a Glc cap (Fig. 4). With the exception of the product of the UDP-Glc: glycoprotein glucosyltransferase (Glc1Man5GlcNAc2) (H6.1), Glc was only added to N-glycans of E. histolytica after the addition of Gal (Fig. 6).

Because the complex N-glycans of E. histolytica that have Glc caps were resistant to all the glycosylhydrolases that we tested, it was not possible to determine whether the Glc-extensions are {alpha}- or β-linked; hence, the configuration of the linkage between Glc and Gal remains to be defined. A pseudo MS/MS/MS experiment performed on B4 ions of Hex8 (which originate from glycans with a linear series of four or more hexoses) suggests the possibility that some of the extending Glc residues are 1,6-linked (supplemental Fig. 4). A full discussion of the mass spectrometry experiments is presented in the supplemental material.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Properties of E. histolytica N-Glycans That Distinguish Them from N-Glycans of Fungi and Metazoa—As shown by analysis of predicted proteins from whole genome sequences, E. histolytica is missing seven asparagine-linked glycosylation enzymes, and so N-glycans are built upon the truncated precursor Man5GlcNAc2 (H5.1 in Fig. 1) (see supplemental Table 1) (3). Man5GlcNAc2 is glucosylated by the UDP-Glc:glycoprotein glucosyltransferase of the N-glycan-dependent quality control system for protein folding to make Glc1Man5GlcNAc2 (H6.1) (4-6). For the same reason, N-glycans of T. vaginalis, the cause of vaginitis, are also built upon Man5GlcNAc2, which is again glucosylated to Glc1Man5GlcNAc2 (3, 6).


Figure 9
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  		FIGURE 9.
MS2 collision-induced dissociation spectrum of the Hex6 GlcNAc2 [M+2Na]2+ isolated at m/z 903. 51. A, the diagram shows the origin of fragments from the isomers present in this pool (see Fig. 2G). Asterisk and pound symbols indicate the origin of cross-ring fragments of the same denomination but different m/z. Diagnostic ions for isomers H6.1 and H6.4 are as follows. Fragments 0,4A5 and 3,5 A5, m/z 301.14 and 329.16 (C) define the unsubstituted Man{alpha}1,6-arm, whereas fragment 1,3A4, m/z 723.35 (E) indicates the four hexoses of the Man{alpha}1,3-arm. Fragment Y3{alpha}/Y3β/B6, m/z 458.20 (B) reveals a biantennary structure. The spectrum supports evidence for Gal extensions. An ion at m/z 315.16 (C) indicates a 1,3A2x fragment. The single isomer predicted to contain terminal Man{alpha}1,2- (H6.2) is just ~1% of the pool; therefore, the majority of the 1,3A2x fragment originates from the terminal Gal{alpha}1,2- of the more abundant isomers H6.3, H6.4, and H6.5. Evidence for extensions on the Man{alpha}1,6- was found as well. Fragments 0,4A4 and 3,5 A4 at m/z 505.19 and 533.21 (D) and fragments 0,4A4 and 3,5 A4 at m/z 709.28 and 737.30 (E) indicate Man{alpha}1,6- with one or two hexoses substitutions. Fragment 1,3A5 at m/z 927.44 (F) indicates an {alpha}1,2 with a four-hexose extension. These data, together with results from glycosylhydrolase digestions, define isomers H6.3, H6.2, and H6.5.

 
In contrast to fungi and metazoa, a large proportion of E. histolytica N-glycans reach the plasma membrane as unmodified Man5GlcNAc2 (H5.1), bypassing ER and Golgi mannosidases and glycosyltransferases. Similarly, a large fraction of mature N-glycans of E. histolytica are composed of Glc1Man5GlcNAc2, as has been described in Leishmania (33). The presence of Glc1Man5GlcNAc2 in mature glycoproteins suggests that it may have other functions in addition to serving as an intermediate in the N-glycan-dependent quality control of folding in the ER lumen (4-6).

Because we did not link N-glycans to sites on particular E. histolytica glycoproteins, there are three possible explanations for the abundance of unmodified Man5GlcNAc2 in the plasma membrane. First, N-glycans on some E. histolytica glycoproteins are never processed. Second, N-glycans at multiple sites on the same glycoprotein are processed differently. Third, E. histolytica mannosidases and glycosyltransferases that make complex N-glycans are very inefficient. We speculate that the high Man N-glycans of E. histolytica may be harder to recognize by the host immune system than the unique complex N-glycans of E. histolytica. A similar argument has been made to explain the abundance of high Man N-glycans on gp120 of HIV (14).

The capping of E. histolytica plasma membrane glycoproteins containing Man5GlcNAc2 by cyanovirin-N suggests the possibility that anti-viral lectins such as cyanovirin-N might be used to block binding of E. histolytica to the host epithelium. Cyanovirin-N inhibits phagocytosis of mucin-coated beads by E. histolytica.3 As well, affinity columns with concanavalin A efficiently capture E. histolytica glycoproteins with N-glycans (so-called N-glycome) for mass spectrometric identification of peptides.3

In addition to OST and UDP-Glc:glycoprotein glucosyltransferase activities (6, 8), the ER of E. histolytica contains a swainsonine-inhibitable {alpha}-mannosidase, which trims a fraction of the N-glycans to Man3GlcNAc2 (H3.1) and Man4GlcNAc2 (H4.1). As in metazoa, the trimmed N-glycans of E. histolytica are building blocks for formation of complex N-glycans (34, 35).

E. histolytica has a single predicted {alpha}-mannosidase (glycosylhydrolase family 92 or GH92), which is similar to those of some fungi (Aspergillus, Neurospora, and Magnaporta) and many Gram-positive bacteria (e.g. Streptococcus, Porphyromonas, Bacteroides, and Mycobacteria) (supplemental Table 1) (20-22). Phylogenetic analyses strongly suggest that the E. histolytica GH92 mannosidase was obtained by lateral gene transfer because the E. histolytica mannosidase is present in a clade with bacteria, and this clade is supported with high bootstrap values (supplemental Fig. 5). In contrast, the fungal mannosidases were present in clades that did not include the E. histolytica mannosidase. Lateral gene transfer is an important mechanism by which E. histolytica has obtained many dozen bacterial genes encoding fermentation and other enzymes (20). E. histolytica is missing mannosidases similar to ER or Golgi mannosidases (GH47) or to lysosomal mannosidases (GH38) (20-22, 32).

The N-glycans of E. histolytica, which have {alpha}1,2-linked Gal that is extended with Glc, are simpler than the complex N-glycans of metazoa. The N-glycans of E. histolytica are consistent with the experimental demonstration of two E. histolytica nucleotide sugar transporters (for UDP-Glc and UDP-Gal) (9) and with bioinformatic predictions of a limited number of Golgi glycosyltransferases in E. histolytica (see supplemental Table 1). O-phosphodiester-linked glycans of E. histolytica are also composed of Gal capped with Glc, but the linkages are different from those of N-glycans (10). Although {alpha}1,2-Gal linked to Man is also present in N-glycans of Schizosaccharomyces pombe (36), N-glycans with distal Glc capping residues are not present in the human host of E. histolytica. This suggests the possibility that the unique complex N-glycans of E. histolytica may be targets for future anti-amebic vaccines. In support of this idea, the O-phosphodiester-linked glycans, which are also rich in Gal and Glc, are one of a limited number of current anti-amebic vaccine targets (1, 2, 10).


   FOOTNOTES
 
* This work was supported, in whole or in part, by National Institutes of Health Grants AI 44070 (to J. S.), GM 43768 (to R. G.), P41 RR10888 and S10 RR15942 (to C. E. C.), and GM 31318 (to P. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1, Figs. 1-5, and additional references. Back

1 To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 715 Albany St., Evans 426, Boston, MA 02118. Tel.: 617-414-1054; Fax: 617-414-1041; E-mail: jsamuels{at}bu.edu.

2 The abbreviations used are: ER, endoplasmic reticulum; MS, mass spectrometry; HPAEC, high performance anion exchange chromatography; PBS, phosphate-buffered saline; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; MES, 4-morpholineethanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Hexn, HexnGlcNAc2; NYT, N{alpha}-acetyl-N-125I-Tyr-Thr-NH2; OST, oligosaccharyltransferase; PNGase F, peptide:N-glycanase F; HPLC, high pressure liquid chromatography. Back

3 D. Ratner and J. Samuelson, unpublished observations. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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D. M. Ratner, J. Cui, M. Steffen, L. L. Moore, P. W. Robbins, and J. Samuelson
Changes in the N-Glycome, Glycoproteins with Asn-Linked Glycans, of Giardia lamblia with Differentiation from Trophozoites to Cysts
Eukaryot. Cell, November 1, 2008; 7(11): 1930 - 1940.
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