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Originally published In Press as doi:10.1074/jbc.M509130200 on August 24, 2005

J. Biol. Chem., Vol. 280, Issue 43, 35929-35942, October 28, 2005
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Deletion of the Glucosidase II Gene in Trypanosoma brucei Reveals Novel N-Glycosylation Mechanisms in the Biosynthesis of Variant Surface Glycoprotein*

Deuan C. Jones1, Angela Mehlert, M. Lucia S. Güther, and Michael A. J. Ferguson2

From the Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom

Received for publication, August 18, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1–3(Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc, Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1–3(GlcNAc{beta}1–2Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc, and Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1–3(Gal{beta}1–4GlcNAc{beta}1–2Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid {alpha}-mannosidase processing was ruled out using a mixture of {alpha}-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man4–3GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The parasitic protozoan Trypanosoma brucei, transmitted by the tsetse fly, is the causative agent of nagana in cattle and African trypanosomiasis or sleeping sickness in humans. During the bloodstream stage of the life cycle, the cells are covered in a densely packed coat of variant surface glycoprotein (VSG)3 The VSG coat serves as a physical barrier to components of the host complement system and undergoes antigenic variation (1). There are many VSG genes, and each encodes a GPI-anchored glycoprotein with one to three N-glycosylation sites (2, 3). The cell line used in this study expresses VSG variant 221 (also known as MiTat1.2). VSG221 carries two occupied N-glycosylation sites, the glycan structures of which have been fully characterized (4). The Asn-428 site, 5 residues from the GPI attachment site, is occupied mostly by oligomannose structures (Man7–9GlcNAc2), whereas the Asn-263 site is occupied by small biantennary structures ranging from Man3GlcNAc2 to Gal2GlcNAc2Man3GlcNAc2.

Protein N-glycosylation serves a wide variety of functions including signaling through interaction with lectins, protein stabilization, protease resistance, endocytic sorting functions, and protein folding (5–7). In eukaryotes, the precursor for N-glycans is built up on the lipid carrier dolichol (Dol) located in the endoplasmic reticulum (ER) membrane. In most organisms, the final precursor is Glc3Man9GlcNAc2-PP-Dol, the glycan portion of which is transferred en bloc via the action of oligosaccharyltransferase (OST), to Asn residues within Asn-X-Ser/Thr sequons during protein translation and sequestration into the lumen of the ER. Processing of the precursor structure by glycosidase and glycosyltransferase enzymes within the ER and Golgi apparatus generates the final set of mature structures. In the first steps of processing, the outer {alpha}1–2-linked glucose is removed by glucosidase I and the two remaining {alpha}1–3 linked glucose residues are removed by glucosidase II. Glucosidase II is a heterodimer; the catalytic {alpha}-subunit is a member of the family 31 glycosyl hydrolases, and the {beta} subunit generally contains a KDEL-type ER retention motif (8, 9). The monoglucosylated Glc1Man9GlcNAc2 structure resulting from removal of the outer two glucose residues allows newly synthesized glycoproteins to interact with the lectin-like "quality control" chaperones calnexin and calreticulin (7, 10, 11). The monoglucosylated structure can also be generated by the re-glucosylation of Man9–7GlcNAc2 structures by UDP-glucose:glycoprotein glucosyltransferase (UGGT), an enzyme that recognizes proteins that are not fully folded (12). Consistent with a role in folding for the monoglucosylated oligosaccharide structures, UGGT shows substrate preference for Man9–7GlcNAc2-containing glycans attached to denatured proteins in vitro (13).

The seminal work of Parodi (for review, see Ref. 14) has shown that protein N-glycosylation in several trypanosomatid parasites is aberrant (15–19). None of these organisms can make Dol-P-Glc and so fail to make glucosylated Dol-PP-oligosaccharide precursors. The mature Dol-PP-oligosaccharide species used for transfer to protein vary according to trypanosomatid species. For example, Trypanosoma conhorini, Trypanosoma dionisii, Leptomonas samueli, Herpetomonas samuelpessoai, and Herpetomonas muscarum utilize triantennary Man9GlcNAc2-PP-Dol; Crithidia fasciculata, Crithidia harmosa, and Leishmania enriettii utilize biantennary Man7GlcNAc2-PP-Dol; Leishmania mexicana, Leishmania adleri, and Blastocrithidia culicus utilize biantennary Man6GlcNAc2-PP-Dol (15–18). Trypanosoma cruzi, the causative agent of Chagas disease in the Americas, utilizes Man9GlcNAc2-PP-Dol during most of its life cycle but uses both Man9GlcNAc2-PP-Dol and Man7GlcNAc2-PP-Dol in its bloodstream trypomastigote stage (19).

The insect-dwelling procyclic form of the African trypanosome T. brucei makes and transfers Man9GlcNAc2-PP-Dol (20). Although bloodstream-form T. brucei also makes Man9GlcNAc2-PP-Dol (21, 22), it was demonstrated in pulse-chase studies by Bangs et al. (23) that one of the two N-glycosylation sites of VSG variant ILTat1.3 receives an Endo H-resistant glycan in the ER. This observation together with the identification by Zamze et al. (4) of unusually small (Man3–4GlcNAc2) N-glycans on mature VSG221 and the accumulation of substantial amounts of Man5GlcNAc2-PP-Dol in bloodstream-form T. brucei (24) suggests two possible models of protein N-glycosylation in the ER of bloodstream-form T. brucei (23), (a) the transfer of Man9GlcNAc2 to some N-glycosylation sites and the transfer of (Endo H-resistant) Man5GlcNAc2 to others and (b) the transfer of Man9GlcNAc2 to both sites and the unusually rapid (immediate) processing of some sites to Endo H-resistant structures in the ER.

To help discriminate between these models and to gain further insights into protein N-glycosylation in this parasite, we constructed a bloodstream-form T. brucei glucosidase II null mutant and used {alpha}-mannosidase inhibitors and assessed their effects on VSG N-glycosylation patterns.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cultivation of Trypanosomes—Bloodstream-form T. brucei genetically modified to express T7 polymerase and the tetracycline repressor protein were cultivated in HMI-9 medium containing 2.5 µg/ml G418 at 37 °C in a 5% CO2 incubator as described in Wirtz et al. (25). In some experiments the parasites were grown for 48 h in the presence of 6 mM 1-deoxynojirimycin (Sigma), the {alpha}-glucosidase inhibitor, or with a mixture of {alpha}-mannosidase inhibitors (186 µM kifunensin (Industrial Research Ltd., New Zealand), 100 µM swainsonine, and 0.8 mM 1-deoxymannojirimycin (Toronto Research Chemicals, North York, Canada)).

Radiolabeling of Trypanosomes and Endoglycosidase Digestions— Cells were washed and resuspended at 2.5 x 107/ml in methionine-free Dulbecco's modified Eagle's medium preincubated with or without 0.8 µg/ml tunicamycin (Calbiochem) for 15 min or with and without the aforementioned mixture of {alpha}-mannosidase inhibitors for 30 min and pulse-labeled with 75 mCi/ml [35S]methionine (Amersham Biosciences) for 3 min at 37 °C. Cells were cooled by the addition of 20 ml of ice-cold trypanosome dilution buffer (26) containing 1 mM methionine and harvested by centrifugation 800 x g for 10 min at 2 °C. The cell pellet was resuspended in 100 µl of 0.5% SDS, 20 mM Tris-HCl, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride, 0.1 mM N{alpha}-p-tosyl-L-lysine chloromethyl ketone, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 0.1 M dithiothreitol, and boiled for 10 min. Aliquots (5 µl) were combined with 20 µl of 50 mM sodium citrate, pH 5.5, containing 0.005 units of Endo H (Roche Applied Science) or with 20 µl of sodium phosphate buffer, pH 7.5, 1% Nonidet P-40 containing 500 units of PNGase F (New England Biolabs). Mock digestion controls were performed with buffer without enzyme. After 3 h at 37°C, samples were mixed with an equal volume of 2x concentrated SDS sample buffer, boiled, and subjected to SDS-PAGE on a 10% gel. The gel was soaked in En3Hance (PerkinElmer Life Sciences) and exposed to Kodak XAR-5 film at –70 °C with a Dupont Cronex intensifying screen.

DNA Isolation and Manipulation—Plasmid DNA was purified from Escherichia coli (DH5{alpha}) using Qiagen Miniprep or Maxiprep kits, as appropriate. Gel extraction and reaction cleanup was performed using Qiaquick kits. Custom oligonucleotides were obtained from Thermo Hybaid or the Dundee University oligonucleotide facility. T. brucei genomic DNA was isolated from ~2 x 108 bloodstream-form cells using DNAzol (Helena Biosciences) or from 5 x 109 procyclic cells using standard methods.

Cloning and Sequencing of the TbGlcaseII{alpha} ORF—A 2517-bp region, including the 2421-bp ORF identified from the T. brucei genome data base, was amplified from genomic DNA by PCR using Pfu DNA polymerase with 5'-tattgttgtttgcttgagtagg-3' and 5'-atgagcaacaagatcagacc-3' as forward and reverse primers, respectively. The cycling parameters used were 95 °C for 5 min, 35 cycles of 95 °C for 45 s, 58 °C for 45 s, and 72 °C for 4 min followed by a final 10-min extension time at 72 °C. The products of three separate PCRs were ligated into the pCR-Blunt II-TOPO vector (Invitrogen), and one representative clone from each was used for triple pass DNA sequencing.

Generation of Gene Replacement Constructs—The 500-bp 5'- and 522-bp 3'-UTR sequences immediately adjacent to the start and stop codons of the TbGlcaseII{alpha} ORF were PCR-amplified from genomic DNA using Taq with 5'-tcaagtacGCGGCCGCtacgttgacggagcgacg-3' and 5'-tggacggtttaaacctaagcgaagcttcactgctagtttttcctactc-3' and 5'-cgcttaggtttaaaccgtccaggatccgatggcgaggccggcgggttg-3' and 5'-tcctcttaGCGGCCGCgacggcgtggaggaatgc-3' as forward and reverse primers, respectively. The two PCR products were used together in a further PCR reaction to yield a product containing the 5'-UTR linked to the 3'-UTR by a short HindIII, PmeI, and BamHI cloning site (underlined) and NotI restriction sites at each end (capital letters). The product was cloned into the pCR4-TOPO vector (Invitrogen) by topoisomerase-mediated ligation. Antibiotic resistance markers were cloned into the HindIII/BamHI restriction sites between the two UTRs to produce two constructs, one containing the puromycin acetyltransferase (PAC) drug resistance gene and one containing the hygromycin phosphotransferase (HPT) hygromycin drug resistance gene.

Transformation of Bloodstream-form T. brucei—Constructs for gene replacement and ectopic expression were purified using the Qiagen Maxiprep kit, digested with NotI to linearize, precipitated, washed twice with 70% ethanol, and redissolved in sterile water. The linearized DNA was electroporated into T. brucei bloodstream cells (strain 427, variant 221) that were stably transformed to express T7 RNA polymerase and the tetracycline repressor protein under G418 selection (25). Cell culture and transformation was carried out as previously described (25, 27).

Southern Blotting—Aliquots of genomic DNA isolated from 100 ml of bloodstream-form T. brucei cultures (~2 x 108 cells) were digested with various restriction enzymes. Fluorescein-labeled probes were generated using the CDP-star random prime labeling kit (Gene Images); 250 ng of template was used in a reaction volume of 50 µl and incubated for 90 min. Aliquots of 8 µl were used for each Southern blot experiment.

Small Scale VSG Isolation—Soluble-form VSG was isolated from 100 ml of cultures containing ~2 x 108 bloodstream-form T. brucei. The cultures were chilled in ice-water and centrifuged at 2500 x g for 10 min. The pellet was washed twice in trypanosome dilution buffer (26) and transferred to a 1.5-ml Eppendorf tube. The pellet was resuspended in 300 µl of lysis buffer (10 mM NaH2PO4 buffer, pH 8.0, containing 0.1 mM 1-chloro-3-tosylamido-7-amino-2-heptanone, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) prewarmed to 37 °C and incubated for 5 min at the same temperature. The sample was centrifuged at 14,000 x g for 5 min, and the supernatant was applied to a 200-µl DE52 anion exchange column preequilibrated in lysis buffer. Fresh lysis buffer (800 µl without protease inhibitors) was applied in four stages, and the pooled column eluate was concentrated and diafiltered with water on a YM-10 spin concentrator (Microcon). The final sample of 50–100 µg sVSG221 was recovered in a volume of 100 µl of water.

Electrospray-Mass Spectrometry (ES-MS) Analysis of Intact VSG— Samples of the small scale VSG preparations were diluted to ~0.05 µg/µl in 50% acetonitrile, 1% formic acid or 50% methanol, 1% formic acid, loaded into nanotips (Micromass type F), and analyzed by positive ion ES-MS on a QTof2 instrument (Micromass) with tip and cone voltages of 1 kV and 30 V, respectively, or an ABI Q-StarXL instrument with tip and declustering potentials of 900 and 60 V, respectively. Data were collected, averaged, and processed using the maximum entropy algorithm using MassLynx software or using the Bayesian protein reconstruction algorithm using ABI Analyst software.

ES-MS/MS Analysis of Pronase Glycopeptides—Samples (50 µl) of the remaining VSG were mixed with 5 µlof1 M ammonium bicarbonate buffer and 10 µl of 10 mg/ml freshly prepared Pronase (Sigma) dissolved in 10 mM calcium acetate. After 48 h at 37 °C, the digest was acidified with 100 µl of 1 M acetic acid, centrifuged in a microcentrifuge, and passed through a small column of 50 µl of Chelex 100 (Na+) over 100 µl of Dowex AG50 (H+). The column was eluted 5 times with 150 µl of water, and the combined eluates, containing the glycopeptides, were dried by rotary evaporation. The samples were dissolved in 50 µlof30% propan-1-ol, 10% acetic acid or 50% methanol, 1% formic acid, and aliquots were loaded into nanotips (Micromass type F) for mass spectrometry. Pronase glycopeptide fractions were analyzed by positive ion ES-MS/MS in neutral loss scanning mode (neutral loss of m/z 81 for the loss of terminal hexose from doubly charged parent ions) using a Micromass Ultima triple quadrupole mass spectrometer. Tip and cone voltages were 1 kV and 40 V, and the collision energy was 15 V with argon as the collision gas at 3 x 10–3 torr. Glycopeptide ions identified in this experiment were analyzed on a QTof2 instrument (using the same source conditions) or on a Q-StarXL instrument, with tip and declustering potentials of 900 V and 60 V, in product ion scanning mode using collision energies of 30–60 V.

Large Scale VSG and VSG N-Glycan Isolation—TbGlcaseII{alpha}–/– null mutant parasites (2 x 1011) were harvested from the infected blood of 20 rats as previously described (27). The parasites were lysed by osmotic shock, and the sVSG was released into the supernatant, purified using DE-52 anion exchange chromatography according to the method of Cross (28). The VSG was further purified by gel filtration on a 850 x 2.5-cm Sephacryl S200 column eluted with 100 mM ammonium bicarbonate at 10 ml/h (29). The protein peak fractions were pooled to yield 36 mg of sVSG after dialysis against water and lyophilization. A sample of sVSG221 (25 mg) was incubated in 200 µl of 0.5% SDS, 1% {beta}-mercaptoethanol (100 °C, 30 min), cooled, and diluted with 800 µl of 125 mM sodium phosphate buffer, pH 7.0, 0.625% Triton X-100, 25 mM EDTA, 1% {beta}-mercaptoethanol. The denatured glycoprotein was then treated with 50 units of PNGase F (Roche Applied Science) for 16 h at 37 °C. The PNGase F digest was made 75% with respect to ice-cold ethanol and incubated at –20 °C overnight to precipitate the bulk of the protein. After centrifugation, the supernatant was dried and dissolved in 100 mM ammonium bicarbonate for gel filtration on a Sephacryl S200 column (13 x 1.5 cm) eluting at 10 ml/h. Fractions of 1 ml were collected, and 10-µl aliquots were spotted onto a silica gel-60 TLC plate (Merck) and stained for carbohydrate with orcinol reagent. The carbohydrate-positive fractions containing the released N-glycans were recovered close to the included volume, well separated from a major A280 absorption peak containing protein and detergent micelles. The glycan-containing fractions were pooled, lyophilized, and desalted by passage through 0.2-ml Chelex 100 (Na+) over 0.5 ml of Dowex AG50 (H+) over 0.5 ml of Dowex AG3 (OH–) over 0.2 ml of QAE-Sephadex A25 (OH–). The eluate and four column washings with 1.4 ml of water were pooled, dried by rotary evaporation, and dissolved in a 100 µl of water.

N-Glycan Fractionation—The N-glycan pool released from sVSG221 was separated by Dionex high pH anion exchange chromatography (HPAEC) on a 4.6 x 250-mm Carbopack PA1 column eluting at 0.6 ml/min with 5 mM sodium acetate in 0.1 M NaOH for 5 min followed by a linear gradient to 150 mM sodium acetate in 0.1 M NaOH over 55 min. Sodium ions were removed from the eluate on-line with a Dionex ARRS unit. Aliquots (1%) of each 0.3-ml fraction were applied to a silica gel-60 TLC plate and stained with orcinol. Carbohydrate-positive fractions were identified eluting between 30.5 and 36.0 min. Aliquots of these fractions (3 µl) were mixed with an equal volume of acetonitrile containing 2% formic acid and analyzed by positive ion nanospray ES-MS to detect [M+2H]2+,[M+H+Na]2+, and [M+2Na]2+ ions of N-glycans. Fractions were pooled according to the ES-MS data. Fraction pools A-E contained predominantly the following structures: Hex8HexNAc2 (A), Hex6HexNAc2 (B), Hex6HexNAc3 (C), Hex7HexNAc3 (D), and Hex9HexNAc2 (E). Each fraction was analyzed by GC-MS monosaccharide composition analysis (30) to estimate yield.

1HNMR—One-dimensional 500-MHz 1H NMR spectra of fractions B and C were obtained using the zgpr pulse program on a Bruker AM500 spectrometer equipped with a 5-mm triple resonance cryoprobe. Samples were repeatedly exchanged into 2H2O, dissolved in 350 µl of 2H2O, and transferred to a Shigemi tube. Experiments were performed at 300 K, with a sweep width of 10 ppm; 256 scans were recorded after 8 dummy scans. Two-dimensional experiments were performed under the same conditions. COSY and ROESY experiments were performed with Watergate suppression, and TOCSY spectroscopy used a phase-sensitive mlev program.

Permethylation and ES-MS/MS of N-Glycans—Samples recovered from NMR were exchanged back into H2O by passage through a short column (20 x 1 cm) of Bio-Gel P-2 (Bio-Rad), equilibrated, and eluted with water. Samples were dried and permethylated by the sodium hydroxide method, as described in Ferguson (30). The permethylated glycans were dissolved in 100 µl of 80% acetonitrile, and aliquots (2 µl) were mixed with an equal volume of 80% acetonitrile, 1 mM sodium acetate before loading into nanotips (Micromass type F) for positive ion ES-MS and ES-MS/MS on a QTof2 mass spectrometer (Micromass, Manchester, UK). Tip and cone voltages were 1 kV and 40 V, respectively, and the collision energy was 45–70 V.

Methylation Linkage Analysis by GC-MS—The remainder of the permethylated glycan samples were subjected to acid hydrolysis, NaB2H4 reduction, and acetylation (to yield partially methylated alditol acetates (PMAAs)), and analyzed by GC-MS as described in Ferguson (30). The PMAAs were analyzed on an Agilent HP-5 column and on a Supelco SP2380 column (the latter to allow resolution of the non-reducing terminal-Man and non-reducing terminal-Glc PMAAs).



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  		FIGURE 1.
Endo H-resistant N-glycans are added to one site of VSG221 in the endoplasmic reticulum. Flourograph of an SDS-PAGE gel of lysates of bloodstream-form T. brucei cells. The cells were pulse-labeled for 3 min with [35S]methionine. Lysates were mock-treated (lanes 1 and 3) or treated with Endo H (lane 2) or PNGase F (lane 4). The apparent molecular weights and glycosylation status of the VSG221 bands are indicated on the left.

 
Tritium Labeling and HPTLC Analysis of Glycans—Samples of pools B, C, and D (2 nmol each) were dried and reduced with 5 µl of 5.5 mM NaB3H4 in 16.6 mM NaOH (16 h, 22 °C). Excess reductant was added (5 µl of 0.5 M NaB3H4), and the incubation was continued for 3 h. The samples were acidified with 10 µl of 1 M acetic acid in a fume hood, passed through 0.2-ml Dowex AG50 (H+), and dried. Boric acid was removed by co-evaporation with 2 x 250 µl of 5% acetic acid in methanol and 2 x 250 µl of methanol. The labeled glycans were dissolved in water and applied to 3-cm-wide strips of Whatman No. 3MM paper for downward paper chromatography in butan-1-ol, ethanol, water (4:1: 0.8). Labeled glycans were recovered from the origin by elution with water and desalted by passage through a column of 100 µl each of Chelex 100 (Na+) over Dowex AG50 (H+) over Dowex AG3 (OH–) over QAE-Sephadex A25 (OH–). The column was eluted 4 times with 400 µl of water, and the combined eluates were dried by rotary evaporation. The 3H-labeled glycans were then purified by Dionex HPAEC, as described above.

The same strategy was used to generate highly purified labeled standards of Man6GlcNAc2 (i.e. Man{alpha}1–6(Man{alpha}1–3)Man{alpha}1–6(Man{alpha}1–2Man{alpha}1–3)Man{beta}1–4GlcNAc{beta}1–4[1-3H]GlcNAc-ol) and NA2 (i.e. Gal{beta}1–4GlcNAc{beta}1–2Man{alpha}1–6(Gal{beta}1–4GlcNAc{beta}1–2Man{alpha}1–3)Man{beta}1–4GlcNAc{beta}1–4[1–3H]GlcNAc-ol). The unlabeled Man6-GlcNAc2 and NA2 glycans were obtained from (Dextra Laboratories, Reading, UK). The Thy-1 GPI standard (i.e. Man{alpha}1–2Man{alpha}1–6(Gal-NAc{beta}1–4)Man{alpha}1–4[1–3H]2,5-anhydromannitol) was prepared from rat brain Thy-1 glycoprotein, as described in Homans et al. (31).

Aliquots equivalent to 10,000 cpm (before and after digestion with jack bean {alpha}-mannosidase (Calbiochem) and E. coli {beta}1–4 galactosidase (Roche Applied Science) or partial acetolysis (32)) were applied to a 10-cm aluminum-backed silica gel-60 HPTLC plate (Merck) and developed with butan-1-ol:ethanol:water (4:3:3 v/v/v) 3 times (32). Radiolabeled components were detected by fluorography at –70 °C after spraying with En3Hance (PerkinElmer Life Sciences) using Kodak X-OMAT AR film and an intensifying screen.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Endo H-resistant N-Glycans Are Added to One Glycosylation Site of VSG221—Variant 221 trypanosomes were pulse-labeled in culture with [35S]methionine for 3 min, and the cell lysates were analyzed by SDS-PAGE and fluorography before and after PNGase F and Endo H treatment. The results show that two forms of VSG are apparent after 3 min of pulse-labeling (Fig. 1, lanes 1 and 3); they are a weakly labeled 53-kDa unglycosylated band (that co-migrates with VSG synthesized in the presence of the N-glycosylation inhibitor tunicamycin; data not shown) and a strongly labeled upper 57-kDa band that co-migrates with mature VSG221 containing 2 occupied N-glycosylation sites at Asn-263 and Asn-428 (4). Treatment with PNGase F, an endoglycosidase that removes all types of N-glycan, converted the upper band to the lower band, as expected (Fig. 1, lanes 4). However, treatment with Endo H, an endoglycosidase that removes only conventional triantennary oligomannose and hybrid N-glycans, converted the upper band to an intermediate 54.5-kDa band representing VSG bearing a single Endo H-resistant N-glycan (Fig. 1, lane 2).

These results show that newly synthesized (ER-resident) VSG221 contains one conventional Endo H-sensitive N-glycan and one Endo H-resistant N-glycan. This reproduces the observation made for a similar doubly N-glycosylated VSG (variant ILtat1.3) (23) and shows that bloodstream-form T. brucei cells exhibit both a conventional and aberrant features of protein N-glycosylation. Thus, whereas in other eukaryotes all N-glycans are Endo H-sensitive until glycoproteins are processed in the Golgi apparatus (several minutes after synthesis in the ER), in T. brucei some N-glycans are Endo H-resistant upon or immediately after transfer to VSG polypeptide in the ER.

Bioinformatic Analysis—Local alignment searching (tBLASTn) (33) of the T. brucei genome data held in GeneDB at The Sanger Institute with yeast and/or mouse genes associated with the biosynthesis of Dol-PP-oligosaccharide donors, their transfer to Asn-X-Ser/Thr sequons, and subsequent ER processing was performed (TABLE ONE). The search using the mouse glucosidase II {alpha} subunit as a query sequence identified two putative open reading frames showing significant sequence similarity (62 and 51%). The homologue with the greatest similarity to the query (gene number Tb10.05.0080) was chosen for further analysis and was later demonstrated to be the T. brucei glucosidase II {alpha} subunit (TbGlcaseII{alpha}). The second homologue (gene number Tb11.01.2140) showed greater similarity to human lysosomal {alpha}-glucosidase and was not studied further. A putative homologue of the human glucosidase II {beta}-subunit was also found (TABLE ONE), suggesting that the T. brucei contains a conventional {alpha}/{beta} glucosidase II assembly in the ER, although the T. brucei {beta}-subunit homologue lacks an identifiable ER retention sequence. Homologues of UGGT and calreticulin were also found (TABLE ONE). However, as in T. cruzi (34) no calnexin homologue was apparent, these data suggest that T. brucei has a relatively conventional eukaryotic calreticulin-mediated glycoprotein refolding quality control system (7, 10, 11). On the other hand, BLAST searches for homologues of eukaryotic genes involved in the biosynthesis of the conventional Glc3Man9GlcNAc2-PP-Dol N-glycan precursor indicated that the ALG6, -8, and -10 genes (encoding the three Dol-P-Glc-dependent {alpha}-glucosyltransferases) were absent as were obvious homologues for Dol-P-Glc synthetase (ALG5) and glucosidase I (TABLE ONE). Similar conclusions were reported recently by Samuelson et al. (35).


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  		TABLE ONE
BLAST search results for T. brucei genes with homology to yeast and mouse genes associated with the dolichol cycle and early events of protein N-glycosylation in the ER

tBLASn searches (33) with yeast (Saccharomyces cerevisiae) and mouse (Mus musculus) protein sequences were used to identify putative T. brucei homologues. The percentages of amino acid identity (ID) and amino acid similarity (Sim) and the bit scores are indicated. EDEM = ER degradation enhancing {alpha}-mannosidase like protein.

 
Cloning and Sequencing of the TbGlcaseII{alpha} ORF—The TbGlcaseII{alpha} ORF was amplified from T. brucei genomic DNA by PCR using Pfu polymerase. Three clones from separate amplifications were sequenced three times each. The consensus sequence (accession number AJ865333 [GenBank] ) revealed 11 base differences compared with the genome data base assembly (gene number Tb10.05.0080). All of the differences were silent with respect to the predicted amino acid sequence and probably represent differences between the strain used for the genome sequencing project (TREU927/4) and that used in this study (strain 427). The amino acid sequence suggests that TbGlcaseII{alpha} encodes a glycosyl hydrolase family 31 protein with a cleavable N-terminal signal peptide for import into the ER (residues 1–19) and two potential N-glycosylation sites at Asn-122 and Asn-736.

Generation of TbGlcaseII{alpha} Null and Conditional Null Mutant Cell Lines—Strain 427 bloodstream-form T. brucei parasites expressing VSG221 and transformed to stably express T7 polymerase and the tetracycline repressor protein under G418 antibiotic selection were used in this study (25) and are referred to here as wild type. Southern blot analysis using a TbGlcaseII{alpha} ORF probe suggested that TbGlcaseII{alpha} is a single-copy gene per haploid genome (data not shown). The genetic modifications described below are summarized in Fig. 2A. Linear DNA containing ~500 bp each of TbGlcaseII{alpha} 5'-UTR and 3'-UTR, surrounding a hygromycin phosphotransferase (HPT) gene, was introduced into bloodstream-form T. brucei parasites by electroporation. Clones in which one allele of TbGlcaseII{alpha} was replaced by HPT by homologous recombination ({Delta}TbGlcaseII{alpha}::HPT) were selected with hygromycin. The second TbGlcaseII allele was replaced in a {Delta}TbGlcaseII{alpha}::HPT clone in the same way with puromycin acetyl-transferase (PAC). Hygromycin and puromycin-resistant clones ({Delta}TbGlcaseII{alpha}::HPT/{Delta}TbGlcaseII{alpha}::PAC null mutants) were selected. Southern blot analysis of the wild-type, {Delta}TbGlcaseII{alpha}::HPT, and {Delta}TbGlcaseII{alpha}::HPT/{Delta}TbGlcaseII{alpha}::PAC null mutant cells with a probe to the TbGlcaseII{alpha} 5'-UTR confirmed that both alleles had been replaced (Fig. 2B). The null mutant cell line had no noticeable differences in gross morphology. However, it exhibited marginally slower growth in culture (doubling time 6.4 ± 0.5 h) than the parental cell line (doubling time 5.5 ± 0.5 h). A tetracycline-inducible ectopic copy of the TbGlcaseII{alpha} gene was introduced into a null mutant clone using the pLew82 vector (25) and phleomycin selection to yield {Delta}TbGlcaseII{alpha}Ti/{Delta}TbGlcaseII{alpha}::HPT/{Delta}TbGlcaseII{alpha}::PAC conditional null mutant clones.



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  		FIGURE 2.
Construction of a TbGlcaII{alpha} null mutant trypanosome. Panel A, summary of the of homologous recombination gene replacement events with HPT and PAC drug resistance genes. Panel B, Southern blot of genomic DNA digested with PstI from wild-type cells (lane 1), {Delta}TbGlcaseII{alpha}::HPT (single-allele replacement) cells (lane 2) and {Delta}TbGlcaseII{alpha}::HPT/{Delta}TbGlcaseII{alpha}::PAC null mutant cells (lane 3). The blot was probed a 5'-UTR probe and indicates the replacement of both alleles with drug resistance genes.

 
Characterization of Intact VSG from Wild Type and TbGlcaseII{alpha} Null and Conditional Null Mutant Cell Lines—The cell lines were grown in vitro, and samples of ~2 x 108 cells from 100 ml of culture were processed to yield 50–100 µg of purified sVSG221. Aliquots were analyzed by positive ion ES-MS. The deconvoluted mass spectra of the intact glycoproteins are shown in Fig. 3. The wild-type profile (Fig. 3A) shows the range of different glycoforms that arise from known heterogeneity in the GPI anchor (36) and N-glycan sites (4) (TABLE TWO). VSG from the TbGlcaseII{alpha} null mutant showed a significant difference in glycoform pattern with a shift of glycoforms to a higher mass equivalent to approximately 3 hexose units (486 Da) (Fig. 3B). This change in glycoform pattern was reversed in the tetracycline-induced conditional null ("add back") mutant (Fig. 3C), demonstrating that the change in VSG glycoform profile was a direct result of TbGlcaseII{alpha} gene deletion. An identical change in VSG glycoforms to that seen in the null mutant was observed when wild-type cells were grown for 48 h in the presence of 6 mM 1-deoxynojirimycin, a broad specificity {alpha}-glucosidase inhibitor (37), before VSG isolation (Fig. 3D). The latter result suggests that the TbGlcaseII{alpha} gene analyzed in this study is the only ER {alpha}-glucosidase involved in N-glycan processing in T. brucei.


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  		TABLE TWO
Isobaric glycoforms of sVSG221 detected by ES-MS

The molecular weights of different glycoforms of sVSG221 were calculated according to the indicated compositions. The -, trace (tr), +, ++, and +++ scores indicate the relative abundances of those glycoforms observed in (Fig. 3) for the sVSG221 preparations from the different cell lines, i.e., wild-type cells, TbGlcase{alpha} null mutant cells (Null mutant), induced conditional null mutant cells (Add-back), and wild-type cells treated with 1-deoxynojirimycin (dNJ).

 



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  		FIGURE 3.
Mass spectrometric analysis of intact sVSG221 from wild-type, TbGlcaII{alpha} null, and 1-deoxynojirimycin-treated trypanosomes. Samples of whole sVSG from small-scale VSG preparations were analyzed by ES-MS on a QTof2 instrument, and the spectra were deconvolved by maximum entropy. The sVSG samples were from wild-type cells (panel A), the TbGlcaII{alpha} null mutant (panel B), the induced TbGlcaII{alpha} conditional null (add back) mutant (panel C), and wild-type cells grown in the presence of the glucosidase inhibitor 1-deoxynojirimycin (dNJ; panel D).

 
Characterization of VSG Pronase Glycopeptides from Wild Type and TbGlcaseII{alpha} Null Mutant Cell Lines—To probe the nature of the changes in VSG glycosylation induced by deletion of the TbGlcaseII{alpha} gene, aliquots of wild-type and null mutant VSG samples (~50 µg) were digested with Pronase and analyzed by ES-MS/MS in neutral loss scanning mode. This method filters the spectrum for doubly charged glycopeptide ions that can lose a terminal hexose residue (m/z 81) when they undergo collision-induced dissociation (Fig. 4, A and B). These data showed no apparent changes in the masses of the GPI-peptide fragments or the C-terminal (Asn-428) Hex9–7HexNAc2-containing glycopeptides. The identities of these ions were confirmed by daughter ion ES-MS/MS on a QTof2 mass spectrometer, e.g. Fig. 4, C and D).

The absence of any hint of Hex10GlcNAc2-NT/NTT ions in (Fig. 4B) is noteworthy. This suggests that deletion of the TbGlcaseII{alpha} gene does not lead to any Glc1Man9GlcNAc2 structures attached to the C-terminal Asn-486 site. On the other hand, the internal (Asn-263) glycopeptides were changed from the previously characterized Hex3HexNac2-, Hex3HexHAc3-, and Hex4HexNAc3-containing species (4) to Hex6HexNAc2-containing species (Fig. 4B). The identities of these ions were confirmed by daughter ion ES-MS/MS on a QTof2 mass spectrometer; e.g. Fig. 4, E and F. To characterize these changes, N-linked glycans were isolated from the null mutant VSG for detailed structural analysis.



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  		FIGURE 4.
Mass spectrometric analysis of Pronase glycoptides from sVSG221 from wild-type and TbGlcaII{alpha} null mutant trypanosomes. Pronase digests of wild-type (panel A) and TbGlcaII{alpha} null mutant (panel B) sVSG221 were analyzed by ES-MS/MS in neutral loss scanning mode (neutral loss of m/z 81) to identify doubly charged glycopeptide ions. The likely identities of the ions, based on measured mass, are indicated. These were further supported by daughter ion scanning ES-MS/MS of individual ions. Representative daughter ion spectra are shown for the [M+2H]2+ Ser-GPI C-terminal glycopeptide ion at m/z 955.7 (panel C), the [M+2H]2+ Hex9HexNAc2-Asn-Thr glycopeptide ion at m/z 1049.7 (panel D), the [M+2H]2+ Hex4GlcNAc3-Asn-Glu-Thr glycopeptide at m/z 810.7 (panel E), and the [M+2H]2+ Hex6HexNAc2-Asn-Glu-Thr glycopeptide ion at m/z 871.1 (panel F). S-EtNP, Serethanolamine phosphate; NE, NET, NT, NTT are single-letter amino acid sequences.

 
Structural Characterization of the N-Linked Glycans of sVSG221 Isolated from TbGlcaseII{alpha} Null Mutant Cells—A large scale VSG221 preparation was made from TbGlcaseII{alpha} null mutant cells. The N-linked glycans were released from 25 mg of sVSG221 using PNGase F and separated by HPAEC. Analysis of the column fractions by ES-MS showed that they eluted between 30.5 and 36.0 min in the order Hex8HexNAc2, Hex6HexNAc2, Hex6HexNAc3, Hex7HexNAc3, and Hex9HexNAc2.

The Hex8HexNAc2 and Hex9HexNAc2 species from the Asn-428 glycosylation site were studied by positive ion ES-MS/MS after permethylation. The daughter ion spectra of the [M+2Na]2+ pseudomolecular ions were identical to those of conventional Man9GlcNAc2 and Man8GlcNAc2 oligomannose glycans (data not shown) and were in agreement with the structures observed previously by Zamze et al. (4). Notably, there was no evidence for the presence of a terminal glucose residue in these structures (that would otherwise give rise to a B-type daughter ion at m/z 853 corresponding to a linear Hex4 branch. To confirm this, aliquots of the native glycans were radiolabeled by reduction in NaB3H4 and analyzed along with radiolabeled oligomannose standards by HPTLC with and without treatment with jack bean {alpha}-mannosidase. Both structures were fully digested to Man{beta}1–4Glc-NAc{beta}1–4GlcNAc-ol, confirming the lack of any jack bean {alpha}-mannosidase-resistant {alpha}Glc residues in the parent structures (data not shown). The novel Hex6HexNAc2 structure from the Asn-263 site was analyzed by GC-MS compositional analysis, one-dimensional and two-dimensional 1H NMR, and after permethylation, ES-MS/MS and GC-MS methylation linkage analysis.

The one-dimensional NMR spectrum revealed eight well resolved anomeric protons. The chemical shift and J1,2 coupling constant values suggested the presence of an {alpha}Glc residue, four {alpha}Man residues, a {beta}Man residue, and two {beta}GlcNAc residues (38) (Fig. 5A). These assignments were confirmed when intra-residue connectivity networks were traced from COSY, ROESY, and total correlation spectra, which allowed assignment of additional protons in each spin system (TABLE THREE). Using the residue descriptors shown in Fig. 5A, inter-residue connectivities were observed in the ROESY spectrum between G3-H1 and D1-H3, D1-H1 and C-H2, and C-H1 and 4-H2 (Fig. 5B). This suggests the linear sequence Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1-. These assignments were consistent with the ES-MS/MS data that show a linear Hex4 branch, indicated by the m/z 853 B-type and m/z 953 Y-type daughter ions (Fig. 6A). The GC-MS methylation linkage analysis showed the presence of a terminal Glc residue together with terminal Man, 3-O-substitiuted Man, 2-O-subsitituted Man, and 3,6-di-O-substituted Man residues (TABLE FOUR). To assess whether the Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man branch was attached to the 3- or the 6-position of the {beta}Man residue of the Man{beta}1–4GlcNAc{beta}1–4GlcNAc unit, the NaB3H4-reduced oligosaccharide was analyzed by HPTLC before and after partial acetolysis. This treatment, which is selective for the cleavage of Man{alpha}1–6Man glycosidic bonds, resulted in the loss of one hexose residue (Fig. 7A, lanes 1 and 2), consistent with a single {alpha}Man attached to the 6-position and the Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man branch attached to the 3-position (Fig. 7C). Taken together, these data provide support for the structure proposed in Fig. 5A. This structure contains a conventional biantennary Man{alpha}1–3(Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc core that is extended on the 3-arm as Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1–3(Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc. The likely origin of this structure is described under "Discussion."


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  		TABLE THREE
Proton NMR assignments for the sugar residues of the Hex6HexNAc2 and Hex6HexNAc3 glycans from Asn-263 of VSG221 from TbGlcasell{alpha} null mutant trypanosomes

Chemical shift assignments for C-H protons of the Hex6HexNAc2 and Hex6HexNAc3 glycans were determined by COSY and total correlation spectroscopy two-dimensional 1H NMR. Correlations were used to assign protons starting from the well resolved H1 anomeric proton region. Figures in brackets belong to the indicated residue spin system but cannot be assigned to specific positions.

 


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  		TABLE FOUR
GC-MS methylation linkage analysis of Hex6HexNAc2 and Hex6HexNAc3 glycans from Asn-263 of VSG221 from TbGlcaseII{alpha} null mutant trypanosomes

The purified Hex6HexNAc2 and Hex6HexNAc3 glycans were permethylated, hydrolyzed, deutero-reduced, and acetylated to yield PMAAs for analysis by GC-MS. Residue types were deduced from the electron-impact mass spectra and retention times. t-Glc and t-Man are non-reducing terminal Glc and Man residues, respectively.

 
Similar analyses of the Hex6HexNAc3 structure showed that this material had an additional terminal {beta}GlcNAc residue on the 6-arm of the Man{alpha}1–3(Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc core. Thus, the NMR data are similar to those for the Hex6HexNAc2 except for the presence of an additional {beta}GlcNAc residue, residue 5' (Fig. 5C) (TABLE FOUR). The ES-MS/MS data again that show a linear Hex4 branch, indicated by the m/z 853 B-type daughter ion together with a non-reducing terminal HexNAc residue that gives rise to the m/z 282 B-type and m/z 1791 Y-type daughter ions (Fig. 7B). Methylation linkage analysis (TABLE 4) also identified this non-reducing terminal GlcNAc residue together with a disappearance of terminal Man and the appearance of additional 2-O-substituted Man. Partial acetolysis of the NaB3H4-reduced Hex6HexNAc3 oligosaccharide produced the same Hex5HexNAc2 product as the NaB3H4-reduced Hex6HexNAc2 oligosaccharide (Fig. 7A, lanes 3 and 4), supporting the notion that the additional {beta}GlcNAc residue is attached to the 4' residue (Fig. 7C). Taken together, these data provide support for the structure proposed in Fig. 5C. This structure contains a conventional biantennary Man{alpha}1–3(Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc core that extended on the 3-arm and the 6-arm as Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1–3(GlcNAc{beta}1–2Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc.



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  		FIGURE 5.
1H NMR analysis of the Hex6HexNAc2 and Hex6HexNAc3 glycans isolated from the Asn-286 site of sVSG221 from TbGlcaII{alpha} null mutant trypanosomes. Panel A, anomeric region of the one-dimensional 1H NMR spectrum of the Hex6HexNAc2 glycan. The H-1 proton resonances were assigned to residue types ({alpha}Glc, {alpha}Man, {beta}Man, {beta}GlcNAc) according to J1,2 coupling constants, chemical shifts, and the chemical shifts of adjacent protons in the sugar rings deduced from two-dimensional COSY spectra (TABLE THREE). Panel B, reporter region of the ROESY spectrum of the Hex6HexNAc2 glycan showing intra- and inter-residue connectivities. The boxed cross-peaks indicate the through-space connectivities that suggest the sequence Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1-. Residue descriptors are those shown in the box in panel A. Panel C, anomeric region of the one-dimensional 1H NMR spectrum of the Hex6HexNAc3 glycan. The H-1 proton resonances were assigned to residue types ({alpha}Glc, {alpha}Man, {beta}Man, {beta}GlcNAc) according to J1,2 coupling constants, chemical shifts, and the chemical shifts of adjacent protons in the sugar rings deduced from two-dimensional COSY spectra (TABLE THREE).

 
There was insufficient Hex7HexNAc3 material for NMR and methylation linkage analysis. However, the m/z 853 B-type daughter ion in the ES-MS/MS spectrum clearly indicates the presence of the linear Hex4 branch together with a terminal Hex-HexNAc unit (indicated by the intense m/z 486 B-type daughter ion) (Fig. 6C). This new terminal Hex residue was deduced to be a {beta}-galactosidase residue in 1–4 linkage to the underlying GlcNAc residue by digestion of the NaB3H4-reduced Hex7HexNAc3 oligosaccharide with E. coli {beta}1–4-specific {beta}-galactosidase (Fig. 7B, lanes 5 and 6). As expected, neither the Hex6HexNAc2 nor the Hex6HexNAc3 was digested by {beta}-galactosidase (Fig. 7B, lanes 1–4). The location of the Gal{beta}1–4GlcNAc unit was inferred by partial acetolysis of the NaB3H4-reduced Hex7HexNAc3 oligosaccharide. Thus, the terminal product of this reaction was also Hex5HexNAc2 (Fig. 7A, lanes 5 and 6), suggesting that it is attached to the 4' residue (Fig. 7C). These data are consistent with the structure Glc{alpha}1–3Man{alpha}1–2Man{alpha}1–2Man{alpha}1–3(Gal{beta}1–4GlcNAc{beta}1–2Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc.



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  		FIGURE 6.
Mass spectrometric analysis of glycans isolated from the Asn-263 site of sVSG221 from TbGlcaII{alpha} null mutant trypanosomes after permethylation. Permethylated glycans were analyzed by daughter ion ES-MS/MS. Panel A, daughter ion spectrum of the [M+2Na]2+-permethylated Hex6HexNAc2 ion. Panel B, daughter ion spectrum of the [M+2Na]2+-permethylated Hex6HexNAc3 ion. Panel C, daughter ion spectrum of the [M+2Na]2+-permethylated Hex7HexNAc3 ion. All spectra were processed (using the MassLynx maximum entropy 3 algorithm) to produce spectra consisting only of singly charged ions.

 
The Effects of {alpha}-Mannosidase Inhibitors on Wild Type VSG221 N-Glycosylation—The novel structures found at the Asn-263 glycosylation site in the glucosidase II null mutant point to the transfer of Man5GlcNAc2, its subsequent glucosylation by UGGT, and processing by GlcNAc- and galactosyl-transferases. However, it is also conceivable that trypanosomes might perform unusually rapid {alpha}-mannosidase trimming of a conventional Man9GlcNAc2 structure to Man5GlcNAc2 at that site.

To address this possibility, we repeated the experiment shown in (Fig. 1) after preincubation with a mixture of cell-permeable {alpha}-mannosidase inhibitors (186 µM kifunensin, 100 µM swainsonine, and 0.8 mM 1-deoxymannojirimycin). The results (Fig. 8A) are very similar to those in Fig. 1; i.e. after a 3-min pulse-label one of the 2 N-glycosylation sites is already endo H-resistant, suggesting that Asn-263 is not rapidly processed by {alpha}-mannosidases from endo H-sensitive Man9GlcNAc2 to endo H-resistant Man5GlcNAc2. To confirm this, we grew wild-type trypanosomes for 48 h in the presence and absence of the {alpha}-mannosidase inhibitor mixture and assessed the effects of the inhibitors on VSG221 N-glycosylation by mass spectrometry. Aliquots of purified VSG221 were analyzed by positive ion ES-MS, and the mass spectra of the intact glycoproteins are shown in (Fig. 8, B and C). The wild-type profile (Fig. 8B) shows the range of different glycoforms that arise from known heterogeneity in the GPI anchor (37) and N-glycan sites (4) (TABLE TWO). VSG from the mannosidase inhibitor-treated cells showed a significant difference in glycoform pattern, with a shift of glycoforms to higher mass (Fig. 8C) and (TABLE TWO). These data indicate that the mannosidase inhibitor mixture is active. To assess the effects of the mannosidase inhibitors on the individual N-glycosylation sites, aliquots of wild-type and mannosidase inhibitor-treated VSG samples were digested with Pronase and analyzed by ES-MS/MS in neutral loss scanning mode, as described earlier. These data showed no apparent changes in the masses of the GPI-peptide fragments but, as expected, a significant reduction in the {alpha}-mannosidase-mediated processing of Man9GlcNA2 to Man8GlcNA2 and Man7GlcNA2 at the C-terminal Asn-428 glycosylation site; compare Fig. 8D with Fig. 8E. The glycans at the internal Asn-263 site were 2 hexose units larger in the {alpha}-mannosidase-treated sample than the wild-type sample; compare Fig. 8D with Fig. 8E. This is consistent with inhibition of the {alpha}-mannosidases responsible for trimming the 3-arm of the biantennary Man5GlcNAc2 to Man3GlcNAc2 but inconsistent with the rapid {alpha}-mannosidase processing of a conventional Man9GlcNAc2 precursor at this glycosylation site.



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  		FIGURE 7.
HPTLC analysis of NaB3H4-reduced glycans from the Asn-263 site of sVSG221 from TbGlcaII{alpha} null mutant trypanosomes. The Hex6HexNAc2, Hex6HexNAc3, and Hex7HexNAc3 glycans were labeled with tritium by NaB3H4-reduction and analyzed by HPTLC and fluorography. The positions of reduced dextran oligomers are indicated on the left. DU, dextran units. Panel A, labeled glycans were analyzed before (–) and after (+) partial acetolysis as indicated to preferentially cleave Man{alpha}1–6Man glycosidic bonds. The interpretation of the intermediate and final products (labeled a–d) are shown in panel C. The control glycan (conventional Man6GlcNAc2) produced intermediate and final products of Man5GlcNAc2 (y) and Man3GlcNAc2 (x), as expected (lanes 7 and 8). Panel B, labeled glycans were analyzed before (–) and after (+) digestion with E. coli {beta}1–4 galactosidase. The interpretation of the product of the digestion of Hex7HexNAc3 is shown inside the dotted box in panel C. The control biantennary glycan (NA2) lost two terminal {beta}Gal residues, as expected (lanes 9 and 10). The control Thy-1 GPI glycan was unaffected by {beta}-galactosidase digestion (lanes 11 and 12), showing that the enzyme was not contaminated with {alpha}-mannosidase or {beta}-N-acetylhexosaminidase activities.

 
Based on the data of Zamze et al. (4) and this paper, the differences in VSG221 glycosylation between wild type, TbGlcaseII{alpha} null mutant trypanosomes, and {alpha}-mannosidase inhibitor-treated wild-type trypanosomes are summarized in Fig. 9.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is clear that, despite slightly retarded growth in the null mutant, TbGlcaseII{alpha} is a non-essential enzyme for the growth of bloodstreamform T. brucei in vitro and in vivo. We were also able to successfully differentiate the null mutant into procyclic form T. brucei in vitro (data not shown), suggesting that the gene is also not required for differentiation to, or survival of the procyclic form of the parasite. In this regard, our results are similar to those of Parodi and co-workers (34, 39), who have shown that, in the related American parasite T. cruzi, other components of the ER glucosylation-dependent quality control system (i.e. calreticulin and UGGT) can be deleted with only moderate effects on parasite growth, differentiation, and infectivity. However, analysis of the biochemical phenotype of the T. brucei glucosidase II null mutant together with BLAST searches for glycosylation gene homologues has helped to reveal and explain some of the idiosyncrasies of protein N-glycosylation in the African trypanosome; summarized in Fig. 10.



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  		FIGURE 8.
The effects of {alpha}-mannosidase inhibitors on VSG221 N-glycosylation. Panel A, fluorograph of an SDS-PAGE gel of lysates of blood-stream-form T. brucei cells. The cells were preincubated with a mixture of {alpha}-mannosidase inhibitors and pulse-labeled for 3 min with [35S]methionine. Lysates were mock-treated (lanes 1 and 3) or treated with Endo H (lane 2) or PNGase F (lane 4). The apparent molecular weights and glycosylation status of the VSG221 bands are indicated on the left. Panels B and C, samples of whole sVSG from untreated (panel B) and {alpha}-mannosidase inhibitor-treated (panel C) trypanosomes were analyzed by ES-MS on a Q-StarXL instrument, and the spectra were deconvolved by Bayesian protein reconstruction. Panels D and E, Pronase digests of sVSG from untreated (panel D) and {alpha}-mannosidase-treated (panel E) trypanosomes were analyzed by ES-MS/MS in neutral loss scanning mode (neutral loss of m/z 81) to identify doubly charged glycopeptide ions. The likely identities of the ions, based on measured mass, are indicated. These assignments were supported by daughter ion scanning ES-MS/MS of individual ions (data not shown). Ions marked with a black triangle belong to Ser-GPI fragments. The N-linked glycopeptides appear as multiple ions due to incomplete Pronase digestion. Thus, the Asn-428 site is found as NT-, NTT-, and TNTT-containing glycopeptides (see panel D), and the Asn-263 site is found as NE- and NET-containing glycopeptides.

 



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  		FIGURE 9.
VSG221 glycosylation in wild-type and TbGlcaseII{alpha} null mutant trypanosomes. The boxed structures show the main N-glycan species found at each N-glycosylation site of mature VSG221 from wild-type (4), TbGlcaseII{alpha} null mutant trypanosomes, and {alpha}-mannosidase inhibitor-treated trypanosmes (this study).

 
Although the T. brucei genome contains homologues of Dol-P-Man synthase and all of the known components needed to synthesize Man5GlcNAc2-PP-Dol, to flip this species into the lumen of the ER and to assemble Man9GlcNAc2-PP-Dol, trypanosome equivalents of the three Dol-P-Glc-dependent {alpha}-glucosyltransferases (encoded by ALG6, ALG8, and ALG10 in yeast) and Dol-P-Glc synthase (ALG5) are absent (TABLE ONE and Samuelson et al. (35)). This is in agreement with published data that show that the largest Dol-PP-oligosaccharide species observed in both bloodstream-form and procyclic-form T. brucei is Man9GlcNAc2-PP-Dol (20–22) and not the Glc3Man9GlcNAc2-PP-Dol species common to most other eukaryotes (7, 10, 11). Perhaps unsurprisingly, a homologue of glucosidase I (that removes the terminal {alpha}Glc residue from Glc3Man9GlcNAc2 protein) is also absent from the genome. On the other hand, there appears to be components of a calreticulin (but not clanexin-)-mediated glycoprotein refolding quality control system, including homologues of UGGT and {alpha}- and {beta}-subunits of glucosidase II. The null mutant generated in this study confirms the biochemical activity of glucosidase II, since in its absence half of the N-linked glycans of VSG are expressed bearing terminal {alpha}Glc residues on their 3-arms. This also infers that the UGGT homologue is expressed in bloodstream-form T. brucei and is enzymatically active.

Although bloodstream-form T. brucei makes Man9GlcNAc2-PP-Dol (21, 22), Bangs et al. (23) have shown that one of the two N-glycosylation sites of VSG variant ILTat.1.3 receives an Endo H-resistant glycan in the ER (23). This observation, reproduced here for VSG221 (Fig. 1), together with the identification by Zamze et al. (4) of unusually small (Man3–4GlcNAc2) N-glycans on Asn-263 of mature VSG221 and the accumulation of substantial amounts of Man5GlcNAc2-PP-Dol in bloodstream-form T. brucei (24), suggests two models of VSG N-glycosylation in the ER of bloodstream-form T. brucei; (a) the transfer of Man9GlcNAc2 to some N-glycosylation sites and the transfer of biantennary (Endo H-resistant) Man5GlcNAc2 to others and (b) the transfer of Man9GlcNAc2 to both sites and the unusually rapid (immediate) processing of some sites to Endo H-resistant structures in the ER. We reasoned, based on the specificity of UGGT for Man9–7GlcNAc2 linked to unfolded protein in other organisms (13), that deletion of the T. brucei glucosidase II would "lock" protein-linked Man9GlcNAc2 structures in a glucosylated form4 and allow us to discriminate between these two models. Thus, we expected to find either according to model (a), Glc1Man9–7GlcNAc2 at the Asn-428 site of VSG221 and unchanged small Endo H-resistant structures at the Asn-263 site or, according to model (b), Glc1Man9–7GlcNAc2 at both sites. However, analysis of the oligosaccharide structures attached to the two N-glycosylation sites of VSG221 in the null mutant revealed unexpected features. Thus, the C-terminal Asn-428 N-glycosylation site was unchanged (carrying predominantly Man9GlcNAc2 and Man8GlcNAc2 chains), whereas the internal Asn-263 site now expressed larger structures due to the presence of an extended ({alpha}Glc-capped) 3-arm (Fig. 9). These results are counterintuitive since the preferred substrate for rat glucosidase II is Glc1Man9GlcNAc2, with Glc1Man5GlcNAc2 being a particularly poor substrate (40). Furthermore, the results suggest that T. brucei UGGT glucosylates Man5GlcNAc2, and not Man9GlcNAc2, on VSG, whereas UGGT from other organisms has been reported to have a marked preference for Man9–7GlcNAc2 over Man5GlcNAc2 (13). These unexpected results prompted us to re-evaluate our assumptions, based on examples from mammalian cells (41), that Man9GlcNAc2 structures attached to protein in the ER of the parasite could not be processed by ER mannosidases to anything other than triantennary endo H-sensitive Man5GlcNAc2 structures. Consequently, we analyzed the effects of a mixture of three potent cell-permeable {alpha}-mannosidase inhibitors (kifunensin, swainsonine, and 1-deoxymannojirimycin), which between them can inhibit all known classes of ER and Golgi {alpha}-mannosidases on the processing of wild-type VSG221 N-glycans. In these experiments (Fig. 8), the inhibitors were clearly active, but the results were consistent with model (a), above.

Thus, we postulate that T. brucei uses two types of dolichol-linked oligosaccharide for transfer to VSG in the ER (Fig. 10); a conventional triantennary Man9GlcNAc2-PP-Dol for transfer to sites destined to remain oligomannose (Man9GlcNAc2 to Man5GlcNAc2) and a biantennary Endo H-resistant Man5GlcNAc2-PP-Dol for transfer to sites destined to be directly involved in calreticulin-mediated protein folding and ultimately to be processed down to Man3GlcNAc2 and thence to complex structures. The fact that this organism uses these mechanisms on different sites of the same glycoprotein suggests that either the different VSG glycosylation sites somehow recruit different Dol-PP-oligosaccharde donors to the same ER translocon-OST complex or that one or both sites are glycosylated after protein translocation into the ER. In the latter model, the different VSG glycosylation sites might then recruit OST complexes with different donor specificities. The fact that post-translational N-glycosylation has been observed for some VSG variants (23, 42) provides tentative support for the latter model.

Although it has been shown that other eukaryotes will transfer structures other than Glc3Man9GlcNAc2 to protein (e.g. many other trypanosomatids transfer Man9GlcNAc2, Man7GlcNAc2, or Man6GlcNAc2 (14), and the protozoan Tetrahymena pyriformis (43) and glucose-starved baby hamster kidney cells (44) transfer Glc3Man5GlcNAc2) this is, to our knowledge, only the second example (after T. cruzi bloodstream trypomastigotes (14, 19)) of a an organism utilizing more than one Dol-PP-oligosaccharide donor under normal growth conditions and the first example of site-specific N-glycosylation in a single glycoprotein.



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  		FIGURE 10.
Summary of the similarities and differences between general eukaryote protein N-glycosylation and processing systems (panel A) and those of bloodstream-form T. brucei (panel B). CNX, calnexin; CRT, calreticulin; EDEM, ER degradation-enhancing {alpha}-mannosidase-like protein; ERAD, ER-associated degradation.

 
In T. brucei, the requirement for such a dual system of protein N-glycosylation may stem from the requirements for rapid of VSG synthesis (10 million copies must be made for each cell division). Thus, once the trypanosomatids lost the strict requirement for glucosylated Dol-PP-oligosaccharides for efficient transfer to protein by OST, they would have also lost the requirement to process down from Man9GlcNAc2 to Man3GlcNAc2 before adding additional sugars. Because, according to precedent (7, 10, 11), the smallest Dol-PP-oligosaccharide to appear in the lumen of the ER is likely to be biantennary Man5GlcNAc2-PP-Dol, it makes metabolic sense that T. brucei should immediately attach this structure to those N-glycosylation sites destined for processing to Man4GlcNAc2,Man3GlcNAc2, and conventional Man3GlcNAc2-based complex structures. Indeed, such a mechanism still allows the transfer and processing of Man9GlcNAc2 to conventional (Man9–5GlcNAc2) oligomannose structures, where these are required, but minimizes the drain on Dol-P-Man donors (necessary for converting Man5GlcNAc2 to Man9GlcNAc2) that are also required for GPI anchor biosynthesis. The latter is prodigious in these organisms because they appear to make a >10-fold excess of GPI precursors over their requirement for VSG anchorage (45).

Finally, it is worth noting that analysis of the VSG of the TbGlcaseII{alpha} null mutant has also revealed significant differences between the parasite and higher eukaryotes in N-glycan processing to complex structures. Whereas in higher eukaryotes the addition of {beta}1–2-linked Glc-NAc residues via GnT-I and GnT-II to the 3- and 6-arms, respectively, of the Man3GlcNAc2 core occurs by GnT-I action on the triantennary acceptor substrate Man5GlcNAc2 followed removal of two {alpha}Man residues and the action of GnT-II on the GlcNAc{beta}1–2Man{alpha}1–3(Man{alpha}1–6)Man{beta}1–4GlcNAc{beta}1–4GlcNAc product (46), this sequence of events does not occur in T. brucei. For example, in the TbGlcaseII{alpha} null mutant it is evident that biantennary Glc1Man5GlcNAc2 can act as a substrate for TbGnT-II (and subsequently for {beta}1–4Gal-T) on the 6-arm. Also, since wild-type trypanosomes normally process the glycan at Asn-263 up to a conventional biantennary complex structure (Fig. 9), it appears that the substrate for TbGnT-I is Man3GlcNAc2 and not triantennary Man5GlcNAc2. We may, therefore, conclude that the Glc-NAc-transferases involved in making complex N-glycans in T. brucei are different from their mammalian counterparts. Indeed, BLAST searches for homologues of the mammalian enzymes fail to return obvious parasite homologues. Such differences in amino acid sequence and substrate specificity point to host/parasite differences in N-glycan processing enzymes that may be exploitable.

In summary, the results presented here support the following conclusions. (i) Bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to VSG in a site-specific manner. (ii) Unlike organisms that exclusively transfer Glc3Man9GlcNAc2, the T. brucei UGGT and glucosidase II enzymes appear to prefer Man5GlcNAc2 Glc1Man5GlcNAc2, respectively, as their substrates (although data from more glycoprotein glycosylation sites are needed to test this hypothesis). (iii) The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man4–3GlcNAc2 or complex structures may have evolved as a mechanism to conserve Dol-P-Man donors for GPI anchor biosynthesis in this organism. (iv) There are fundamental differences in the acceptor substrate specificities of host and parasite Glc-NAc-transferases that initiate the synthesis of complex N-glycans.


   FOOTNOTES
 
* This work was supported in part by Wellcome Trust Programme Grant 071463. The costs of publication of this article were defrayed in part by the payment of page charges. This article therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by a Ph.D. studentship from the Medical Research Council. Back

2 To whom correspondence should be addressed: Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK. Tel.: 44-1382-344219; Fax: 44-1382-348896; E-mail: m.a.j.ferguson{at}dundee.ac.uk.

3 The abbreviations used are: VSG, variant surface glycoprotein; sVSG, soluble-form VSG; Dol, dolichol; Dol-P, dolichol phosphate; Dol-PP, dolichol pyrophosphate; Endo H, endoglycosidase H; ER, endoplasmic reticulum; ES-MS, electrospray-mass spectrometry; ES-MS/MS, electrospray-tandem mass spectrometry; GnT, GlcNAc-transferase; GPI, glycosylphosphatidylinositol; HPAEC, high pH anion exchange chromatography; HPT, hygromycin phosphotransferase; HPTLC, high performance thin layer chromatography; OST, oligosaccharyltransferase; PAC, puromycin acetyltransferase; PMAA, partially methylated alditol acetate; PNGase F, peptide N-glycosidase F; UGGT, UDP-Glc:glycoprotein glucosyltransferase; ROESY, rotating-frame Overhauser effect spectroscopy; ORF, open reading frame; UTR, untranslated region. Back

4 No homologue for ER endomannosidase was found in the T. brucei database. Back


   ACKNOWLEDGMENTS
 
We thank Annette Herscovics for advice on mannosidases, Jay Bangs for helpful discussions, David Norman for help with NMR, and the reviewer of an earlier version of this paper for excellent suggestions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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