Unique Asn-linked Oligosaccharides of the Human Pathogen <em>Entamoeba histolytica</em>^{<a href="#fn1" rid="fn1" class=" fn">*</a>}^{<a href="#fn2" rid="fn2" class=" fn">S⃞</a>}

EXPERIMENTAL PROCEDURES

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-^{H]Man (30 Ci/mmol), [U-C]Glc (200 mCi/mmol), UDP-[H]Gal (5.8 Ci/mmol), and UDP-[H]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 α-mannosidase and almond β-glucosidase were from Sigma. Coffee bean α-galactosidase, β1,3,4,6-galactosidase, and β-glycosidase II (β-galactosidase and β-glucosidase activities) were from Calbiochem. Aspergillus saitoi α1,2-mannosidase, bovine testes β-galactosidase, Jack bean hexosaminidase, Man₃GlcNAc₂, and Man₁GlcNAc₂ standards were from Glyko (San Leandro, CA). Maltase and amyloglucosidase (α-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).

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 cm ^{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-H]Man or 2 mCi of [C]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-[^{H]Gal and UDP-[C]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, [^{H]Glc was incorporated into} E. histolytica glycoproteins when labeling with UDP-[^{H]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 m}m 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 × 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 × 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).

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 × 10 ^{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 × 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 (K_d = (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 Δalg3 Saccharomyces cerevisiae (4)) were used to calibrate the system. For non-radiolabeled samples, peaks were collected according to the K_d 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 × 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 Δalg3 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 × 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 × 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-^{H]Man, [H]GlcN, [H]Glc 1-phosphate, and UDP-[H]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 α-galactosidase, maltase, and amyloglucosidase; 100 mm sodium acetate buffer, pH 4.8, was used for Jack bean α-mannosidase, β-glucosidase, β1,3,4,6-galactosidase, α1,2-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.

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 × 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 μmN^-acetyl-N-^I-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 ×100 primary magnification and deconvolved using Applied Precision softWoRx software.

RESULTS

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-^{H]Man (shown in} Fig. 2) or [U-^{C]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. ​Figs.88 and ​and9).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).

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.

FIGURE 2.

The complexity of N-glycans made by E. histolytica trophozoites increases dramatically with time.E. histolytica trophozoites were incubated with [2-^{H]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 Hex_n 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.

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-^{C]Glc and separated by Bio-Gel P-4 filtration. The number of hexoses in each glycomer is indicated (Hex}_n), according to calibrated standards prepared from Man₅GlcNAc₂ digested with jack bean α-mannosidase and A. saitoi α1,2-mannosidase. B, relative abundance (weight percentage) of neutral sugars for each peak after acid hydrolysis, as determined by HPAEC chromatography. fract., fraction.

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 Manα1,6-arm indicates what corresponds to the residual fragment 11.5, which was fully characterized in a separate [U-^{C]Glc-labeling experiment. Note that jack bean α-mannosidase (}JBAM) removes an exposed Manα1,3- (as in isomer H4.3), but it does not remove the Manα1,6-unless the Manα1,3-arm is digested first. Hence, isomer H4.2 becomes susceptible to jack beanα-mannosidase digestion only after removal of Galα1,2- by α-galactosidase. The mode of action of jack bean α-mannosidase (37) supports our assumption that the different sensitivities of H4.2 and H4.3 are due to accessibility of α-linked Man residues. In accordance with this, the underlying structures of other isomers were inferred as well. ASAM, A. saitoi α1,2-mannosidase.

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 α1,2-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).

FIGURE 6.

Synthesis of complex N-glycans in vitro by incubating E. histolytica membranes with UDP-[^{H]Glc and UDP[H]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 α-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 α-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 α-galactosidase. In contrast, Glc₁Gal₁Man₃GlcNAC₂ (H5.6 at 19 min) is not digested by α-galactosidase. G, the table summarizes these results. Isomer H5.2 was fully characterized using complementary data acquired in a separate [2^{H]Man-labeling experiment (}Fig. 2). frct., fraction; Endomann, endomannosidase; comp, composition; ident, identity.

FIGURE 7.

E. histolytica membranes contain a swainsonine-sensitive α-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, Man_3-9GlcNAc₂-NYT.

FIGURE 8.

MS ^{collision-induced dissociation spectrum of the Hex}₃HexNAc₂ 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, Y_3α/Y_3β/B₃ at m/z 458.21 (B), is consistent with archetypal biantennary Man₃GlcNAc₂. Cross-ring fragments ^A₂, m/z 301.14 and ^A₂, m/z 329.16 (C) define the Manα1,6-arm. A low abundance ^A₂ fragment at m/z 315.16 (C) indicates the presence of an isomer bearing a terminal 1,2-linked Gal (H3.2). A Y₃/^A₄ 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 Manα1,6-). Therefore, the structure for H3.2 indicates a missing Manα1,6-arm, with the remaining Manα1,3-arm extended with Galα1,2-. These results are consistent with the glycosylhydrolase digestion data (Fig. 4).

FIGURE 9.

MS ^{collision-induced dissociation spectrum of the Hex}₆ GlcNAc₂ [M+2Na] ^{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 ^A₅ and ^A_5,m/z 301.14 and 329.16 (C) define the unsubstituted Manα1,6-arm, whereas fragment ^A_4,m/z 723.35 (E) indicates the four hexoses of the Manα1,3-arm. Fragment Y_3α/Y_3β/B_6,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 ^A_2x fragment. The single isomer predicted to contain terminal Manα1,2- (H6.2) is just ∼1% of the pool; therefore, the majority of the ^A_2x fragment originates from the terminal Galα1,2- of the more abundant isomers H6.3, H6.4, and H6.5. Evidence for extensions on the Manα1,6- was found as well. Fragments ^A₄ and ^A₄ at m/z 505.19 and 533.21 (D) and fragments ^A₄ and ^A₄ at m/z 709.28 and 737.30 (E) indicate Manα1,6- with one or two hexoses substitutions. Fragment ^A₅ at m/z 927.44 (F) indicates an α1,2 with a four-hexose extension. These data, together with results from glycosylhydrolase digestions, define isomers H6.3, H6.2, and H6.5.

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 [^{H]Man or for 3 h with [C]Glc (Figs.} ​(Figs.22 and ​and3)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-^{H]Man, washed, and chased for 3 h with unlabeled Man, biosynthetic Man}₅GlcNAc₂ 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 Manα1,2-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-^{H]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-[^{H]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 α-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 [^{H]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).

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 α-Linked Gal to Either Arm of Man_3-5GlcNAc₂—E. histolytica membranes incubated with UDP-[^{H]Gal or UDP-[H]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 [^{C]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.

As shown by sensitivity to α-galactosidase but not to β-galactosidase, a single α-linked Gal was added to the terminal Man on either or both arms of Man_2-5GlcNAc₂ 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. ​(Figs.33 and ​and4). 4). Fig. 8B shows the signature of a terminal 1,2-linked hexose (a ^A₂ fragment that cannot originate from Man₃GlcNAc₂) (H3.1). This result indicates that the minor Hex₃ isomer (H3.2) is indeed elongated with an α1,2-linked Gal. The spectrum for Hex₆N-glycans (Fig. 9) also shows the cross-ring ^A₂ 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-^{H]Man (}Fig. 2) or [^{C]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 α- 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 α- 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

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).

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 α-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 α-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 α1,2-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 α1,2-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).

Supplementary Material