The MPB83 Antigen from Mycobacterium bovis Contains O-Linked Mannose and (1 right-arrow  3)-Mannobiose Moieties*

Stephen L. MichellDagger §, Adam O. WhelanDagger , Paul R. WheelerDagger , Maria Panico, Richard L. Easton, A. Tony Etienne, Stuart M. Haslam, Anne Dell||, Howard R. Morris**, Andrew J. Reason**, Jean Louis HerrmannDagger Dagger , Douglas B. Young§§, and R. Glyn HewinsonDagger

From the Dagger  TB Research Group, Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, United Kingdom, the  Department of Biological Sciences, Imperial College of Science Technology and Medicine, London SW7 2AY, United Kingdom, ** M-Scan Mass Spectrometry Research and Training Centre, Silwood Park, Ascot SL5 7PZ, United Kingdom, the Dagger Dagger  Service de Microbiologie, Hopital Saint Louis, 1 Avenue Claude Vellefaux, 75475 Paris, Cedex 10, France, and the §§ Centre for Molecular Microbiology and Infection, Imperial College of Science Technology and Medicine, London SW7 2AY, United Kingdom

Received for publication, August 5, 2002, and in revised form, January 6, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins, although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone of a second mycobacterial glycoprotein, antigen MPB83 from M. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were joined by a (1 right-arrow 3) linkage, in contrast to the (1 right-arrow 2) linkage previously observed in antigen MPT32 from M. tuberculosis and the (1 right-arrow 2) and (1 right-arrow 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and elucidating the mechanisms of mycobacterial glycosylation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein glycosylation is ubiquitous in eukaryotes and the diverse array of carbohydrate moieties that can be fashioned to a polypeptide backbone makes glycoproteins ideal molecules to allow specific interactions with other molecules. In contrast, the number of reports of this post-translational modification by prokaryotes is comparatively low. Examples of eubacterial glycoproteins identified to date include cell surface or secreted proteins of pathogens that have antigenic properties and play a role in host pathogen interaction (1-6). Glycosylation of pilin has been postulated to enhance the adherence of Neisseria meningitidis to endothelial cells (2), whereas N-glycosylation of the platelet aggregation-associated protein of Staphylococcus sanguis may promote colonization of the endocardium (7, 8). Removal of a sero-specific glycosyl moiety from the flagellin of Campylobacter jejuni has been reported to alter the O antigenicity of the organism (9, 10). Similarly, Romain et al. (11) demonstrated that removal of covalently bound mannose from the Mycobacterium tuberculosis antigen MPT32 reduced by 10-fold its ability to elicit a delayed-type hypersensitivity reaction in guinea pigs immunized with Mycobacterium bovis BCG.

M. tuberculosis and M. bovis are closely related members of the "M. tuberculosis complex" (MTb complex) that secrete a series of immunodominant antigens that have been reported to be glycosylated, on the basis of their ability to bind lectins such as concanavalin A (ConA)1 (12-15). Structural confirmation of protein glycosylation by mycobacteria was provided by detailed chemical compositional analysis of MPT32, a 45/47-kDa secreted antigen of M. tuberculosis (15, 16). Mannose, mannobiose, and mannotriose substituents were found O-linked to four threonine residues on the protein backbone. Also, site-directed mutagenesis of the 19-kDa lipoprotein antigen of M. tuberculosis implicated threonine residues in ConA binding, consistent with the observed O-linked glycosylation (17).

On the basis of monoclonal antibody-binding studies, Fifis et al. (13) proposed that the 25/23-kDa secreted antigen of M. bovis was a glycosylated form of the major M. bovis antigen MPB70. They suggested that the increases in molecular weight, relative to the 22,000 MPB70, were attributable to glycosylation with mannose, as treatment of these antigens with alpha -mannosidase resulted in a reduction in their relative molecular weight. Subsequently, we demonstrated the presence of a gene, mpb83, which encodes a protein with 61% identity at the amino acid level to MPB70 (18). In the same study, the protein encoded for by this gene, MPB83, was shown to bind a monoclonal antibody specific for the 25/23-kDa M. bovis antigen. Thus, the 25/23-kDa antigen characterized by Fifis et al. (13) is MPB83, rather than a glycosylated form of MPB70.

M. bovis expresses high levels of both MPB70 and MPB83 and these proteins are strongly recognized by the immune system during M. bovis infection in cattle and badgers (19, 20). Although the proteins are expressed only at low levels in M. tuberculosis when grown in vitro, they are highly immunogenic during infection with live bacteria in mice (18) and man (21). In addition, vaccination of mice with a plasmid encoding MPB83 has been shown to confer significant protection against challenge with M. bovis (22). Thus, given its strong recognition by the immune system, further characterization of MPB83 is required. Open reading frames with homology to MPB70 and MPB83 can be identified in genome sequences from Streptomyces coelicolor and other microbes, but a physiological function has yet to be ascribed to these proteins.

The aims of this study were to explore the glycosylation status of MPB83 and MPB70, to map specific sites for glycosylation within the molecules, and to elucidate the chemical structure of the carbohydrate moiety. Using a combination of genetic and biochemical approaches we have demonstrated that MPB83 but not MPB70 is glycosylated, and that glycosylation occurs via attachment of short (1right-arrow3)-linked mannose chains to two threonine residues.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains and Culture Conditions-- Escherichia coli DH5alpha (Invitrogen) and Mycobacterium smegmatis mc2155 expressing PhoA hybrid proteins were grown in Luria broth, 50 µg/ml hygromycin, or in Middlebrook 7H9 supplemented with albumin, dextrose, catalase (MADC), and 0.05% Tween 80, 200 µg/ml hygromycin, respectively. Alkaline phosphatase activity of E. coli and mycobacterial PhoA recombinants was determined by the presence of blue colonies when plated on L agar (containing appropriate hygromycin concentrations) supplemented with 40 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine (Sigma).

Oligonucleotides and PCR Amplifications-- The genes encoding MPB70, MPB83, and those regions of MPB83 used for deletion mapping, were amplified by PCR using the oligonucleotides described in Table I. Oligonucleotides were supplied by Oswel, Southampton, United Kingdom. PCR amplifications were carried out in a DNA thermal cycler (Biometra) using 0.5 units of cloned Pfu DNA polymerase (Stratagene). PCR reactions were performed in 50-µl volumes containing: 50 mM-KCl, 10 mM Tris-HCl (pH 8.8), 0.01% (w/v) gelatin, 5 mM MgCl2, 200 µM of each dinucleotide triphosphate (dNTP), and 20 pmol of each oligonucleotide primer overlaid with mineral oil. The parameters for amplification were one denaturation cycle at 96 °C for 2 min followed by 30 cycles of denaturation at 96 °C for 1.5 min, hybridization at 56 °C for 1.5 min, and elongation at 72 °C for 1.5 min. A final extension at 72 °C for 5 min was also performed.


                              
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Table I
Oligonucleotides used for fragment amplification
The restriction endonuclease sites are underlined and the site-directed mutations are shown in bold.

Construction of PhoA Hybrids-- mpb70 and mpb83 were amplified from the cosmid pA3, described previously (18), using the oligonucleotide pairs 1,2 and 3,4, respectively (Table I). Amplified fragments were purified using Wizard PCR Prep columns (Promega) and digested with the restriction endonucleases BamHI and KpnI (Promega). Digested fragments were cloned into BamHI/KpnI-digested p19pro-PhoA (Fig. 1), a modified version of pSMT3(19-PhoA) (17) containing a leaderless E. coli alkaline phosphatase (phoA) gene, using standard procedures (Sambrook et al. (23)). p19proSP-PhoA contains the promoter and signal peptide of the 19 kDa of M. tuberculosis in-frame with the PhoA gene in 19pro-PhoA and is used as a positive control for anti-alkaline phosphatase antibody activity and as a negative control for ConA binding. Truncated versions of mpb83 were amplified using oligonucleotide 83PF with the corresponding reverse complementary oligonucleotide and cloned into p19pro-PhoA as described above. Recombinants with functional alkaline phosphatase activity were selected after transformation into competent E. coli DH5alpha (Invitrogen), by the presence of blue colonies when grown on L-agar supplemented with 40 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine. Plasmid DNA was isolated (Qiagen) and subsequently transformed into M. smegmatis mc2155 according to the method of Snapper et al. (24).

Site-directed Mutagenesis-- Complementary forward and reverse oligonucleotide primers 9 and 10 (Table I) were used in conjunction with the reverse complementary and forward oligonucleotide primers, 4 and 3 (Table I), for mpb83, respectively. Following purification, the two products, having an overlap of 30 bp, were combined and amplified by PCR using oligonucleotide primers 3 and 8 to give a fragment of mpb83 incorporating the desired mutation. All cloned DNA fragments were DNA sequenced using the TaqFS dideoxy terminator cycle sequencing kit in conjunction with a 373A Automated DNA Sequencer (Applied Biosystems Inc.). Each nucleotide was sequenced a minimum of three times from both strands. DNA sequence data analysis and protein sequence comparison were performed using the DNASTAR software package (DNASTAR Inc.).

SDS-PAGE and Western Blotting-- For SDS-PAGE, recombinant M. smegmatis was grown in 40 ml of MADC-Tween 80 at 37 °C, 225 rpm, for 48 h. The mycobacteria were pelleted by centrifugation (4,000 rpm for 10 min, Sigma 3K10, rotor 11133), washed twice with 40 ml of PBS (pH 7.0), and resuspended in 3 ml of PBS (pH 7.0). The resulting suspensions were sonicated on ice 10 times for 60 s with 90-s intervals, using a Soniprep 150 (MSE Ltd) equipped with a 1-cm probe. Sonicated extracts were centrifuged at 14,000 rpm for 30 min at 4 °C, the supernatant was isolated and filtered through a 0.22-µm filter. Total protein concentrations of sonicated extracts were determined using the bicinchoninic acid (BCA) protein assay (Pierce and Warriner). 10 µg of extracts were solubilized by heating at 100 °C for 3 min in an equal volume of sample loading buffer {5 mM Tris-HCl, pH 6.8, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, 0.002% (w/v) bromphenol blue). Samples were fractionated by electrophoresis through 12.5% acrylamide (w/v) SDS-polyacrylamide gels using a discontinuous Tris-HCl buffer system as described by Laemmli (25) and transferred to nitrocellulose by electroblotting as described previously (26). Rainbow protein markers (Amersham Biosciences) were run as molecular mass standards.

ConA Analysis and Antibody Detection-- ConA binding was determined by first blocking the nitrocellulose membranes with 20 ml of 5% bovine serum albumin (Sigma) in PBS with 0.1% Tween 20 (Bio-Rad) for 1 h at room temperature. After washing the membranes three times for 10 min with PBS containing 0.5% Tween 20, they were incubated with 20 ml of peroxidase-conjugated ConA at 1 mg/ml in PBS, 0.5% Tween 20 without bovine serum albumin for 1 h at room temperature. The membranes were then washed twice for 10 min with PBS containing 0.5% Tween 20 and finally for 10 min with PBS only. The substrate solution for visualizing peroxidase activity was prepared by dissolving 30 mg of 4-chloronaphthol (Sigma) in 12 ml of methanol followed by addition of 96 ml of PBS and 17 µl of 30% (v/v) hydrogen peroxide. For detecting PhoA protein expression or MPB83, the membranes were blocked with 0.01 M Tris-buffered saline (TBS) containing 3% (w/v) skimmed milk powder (TBSM), and then incubated for 1 h at room temperature. Following three 10-min washes with TBS containing 0.05% (v/v) Tween 20 (TBST) the nitrocellulose membranes were incubated for 2 h at 37 °C with monoclonal antibody raised against bacterial alkaline phosphatase (Sigma) or MBS43 (VLA Weybridge), both at a dilution of 1 in 8000 in 30 ml of TBST containing 3% (w/v) skimmed milk (TBSTM). Membranes were then washed in TBST as described previously and incubated for 2 h at 37 °C with alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) diluted 1 in 8000 in TBSTM. After three further 10-min washes at room temperature with TBS bound alkaline phosphatase-conjugated antibody was detected with SigmaFast tablets (Sigma) dissolved in 15 ml of water.

Purification of Native MPB83-- Native MPB83 was purified from a M. bovis culture supernatant. M. bovis AN5 was grown as a surface pellicle on Bureau of Animal Industry medium (14 g of L-asparagine, 1.5 g of dipotassium hydrogen phosphate, 0.74 g of sodium citrate, 1.5 g of magnesium sulfate, 0.3 g of ferric sulfate, 0.08 g of zinc sulfate, 0.008 g of manganese chloride, 0.00138 g of cobaltous chloride, 10 g of glucose, 100 g of glycerol/liter) for 10 weeks at 37 °C. The culture was filtered through a stainless steel mesh strainer and then passed through a 0.2-µm VacuCap bottle top filter (Gelman Sciences). The culture supernatant was filtered a second time though a 0.2-µm VacuCap bottle top filter to ensure sterility.

The culture supernatant was diluted 3-fold in preparative anion exchange chromatography (AEC) loading buffer (20 mM Tris, pH 8, 0.05% (v/v) Igepal CA-630) and applied to a XK50/20 column (Amersham Biosciences) containing 200 ml of Fast Flow DEAE anion exchange medium (Amersham Biosciences) using a fast protein liquid chromatography instrument. Adsorbed protein was eluted from the column by applying a linear increasing gradient of 0-250 mM sodium chloride over a 1-liter volume. Fractions containing MPB83 were identified by separation on SDS-PAGE gels, transfer to nitrocellulose, and probing with the MPB83-specific antibody MBS43 using a Western blotting procedure. The purity of each MPB83 containing fraction was assessed by total protein analysis of SDS-PAGE gels using a silver stain procedure. Fractions containing MPB83 were pooled and concentrated using a 50-ml stirred cell concentrator containing a YM10 membrane (10,000 molecular weight cut-off) (Millipore UK).

The enriched MPB83 containing material was further purified by high resolution AEC using a MonoQ HR10/10 column. The sample was diluted 5-fold in AEC loading buffer (20 mM Tris, pH 8) and applied to a MonoQ HR10/10 column using a fast protein liquid chromatography instrument. The adsorbed material was eluted from the column by applying a linear increasing gradient of 0-250 mM sodium chloride over a 100-ml volume. Fractions containing MPB83 were identified as described above. The pooled fractions were concentrated by ultrafiltration using a CentriPrep 10 (10,000 molecular weight cut-off) concentrator unit (Millipore UK) centrifuged at 2500 × g (MSE Mistrial 2000 centrifuge, Sanyo Gallenkamp).

The AEC purified material was diluted 5-fold in hydrophobic interaction chromatography loading buffer (20 mM sodium phosphate, pH 7, 1 M ammonium sulfate) and applied to a HR5/5 phenyl-Superose hydrophobic interaction chromatography column (Amersham Biosciences) using a fast protein liquid chromatography instrument. Adsorbed material was eluted by applying a linear, decreasing gradient, of 1-0 M ammonium sulfate over a volume of 80 ml. Fractions containing MPB83 were identified as described above. The pooled fractions were concentrated by ultrafiltration using Centricon 10 (10,000 Mr cut-off) concentrator units (Millipore UK) centrifuged at 4000 × g (MSE Mistral 2000 centrifuge, Sanyo Gallenkamp). The concentrated material was dialyzed against 4× 2 liters of PBS using a 3,500 Mr cut-off dialysis cassette (Perbio Science). Dialysis was performed at 4 °C and a minimum of 2 h was allowed between each change of dialysis buffer. The dialyzed material was filtered through a 0.2-µm syringe filter and the protein concentration was determined using the BCA protein estimation assay.

Chemical Deglycosylation of Native MPB83-- Purified native MPB83 was subjected to a chemical deglycosylation procedure using a GlycoFree deglycosylation kit (Glyko Inc.). This method employs the use of anhydrous trifluoromethanesulfonic acid, which cleaves N- and O-linked glycans nonselectively from glycoproteins while leaving the primary structure of the protein intact (27). Purified native MPB83 was initially desalted by dialysis against 4× 2-liter volumes of water using a 3,500 Mr cut-off dialysis cassette (Perbio Science). The desalted sample was then frozen at -80 °C and lyophilized overnight in a model EF03 freeze dryer (Edwards High Vacuum International). The deglycosylation procedure was then performed in accordance with the GlycoFree kit recommendations.

Briefly, a reaction vial, containing 0.5 mg of lyophilized material, was placed in a dry ice/ethanol bath and 50 µl of trifluoromethanesulfonic acid was slowly added. The vial was then incubated at -20 °C for 4 h and then returned to a dry ice/ethanol bath, to which 150 µl of pyridine was slowly added. The vial was then transferred to dry ice for a further 5 min and then to wet ice for a further 15 min. The reaction mixture was neutralized by addition of 400 µl of 0.5% (w/v) ammonium bicarbonate. The deglycosylated protein was then isolated from the reaction products and reagents by dialysis against 4× 2 liters of PBS using a 3,500 Mr cut-off dialysis cassette (Perbio Science). The protein concentration of the dialyzed material was determined using the BCA protein estimation assay.

Nanoelectrospray Mass Spectrometry-- Intact MPB83 and products of tryptic digestion were analyzed by ES-MS using a nanospray ion source on a hybrid quadrupole orthogonal acceleration time of flight (Q-TOF) mass spectrometer (Micromass UK) (28, 29). Signals attributable to glycopeptides were passed separately for collisionally activated decomposition tandem MS experiments (CAD MS/MS) into a collision cell filled with argon gas, using collision energies from 10 to 40 eV as appropriate (28, 29).

Carbohydrate Analysis-- The modifying sugars of purified MPB83 were identified by first hydrolyzing the protein (50 µg) with 4 M trifluoroacetic acid at 95 °C for 4 h to release the sugars. The hydrolysis reaction was dried under vacuum at 40 °C in a SCV100H SpeedVac apparatus (Stratech Scientific) and then resuspended in 100 µl of water. A sample of 10 µl was then analyzed on a CarboPak PA1 anion exchange column (Dionex Corp.) using a 625LC high performance liquid chromatography instrument (Waters) equipped with a pulsed amperometric detector (Dionex Corp.).

Sugars were eluted from the column with 16 mM sodium hydroxide at a flow rate of 1 ml/min. Sugars were identified by comparing the column retention time with that of sugar standards prepared at known concentrations run under identical conditions (all sugar standards were supplied by Sigma).

FAB-MS and GC-MS Linkage Analysis-- Reductive elimination, permethylation, preparation of partially methylated alditol acetates, and acquisition of FAB-MS and GC-MS data were performed as described previously (30, 31).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant MPB83 but Not MPB70 Is Glycosylated by M. smegmatis-- A recombinant mycobacterial expression system (17, 32) was used to determine whether MPB70 and MPB83 were glycosylated by mycobacteria. The genes encoding these proteins were cloned in-frame with E. coli alkaline phosphatase (PhoA) lacking its own signal sequence in the mycobacterial shuttle vector 19pro-PhoA (Fig. 1) as described under "Experimental Procedures." PhoA was used as a hybrid partner to allow the level of expression of the different constructs to be monitored and to verify that the fusion proteins were being exported. The fusion proteins encoded by these constructs expressed functionally active alkaline phosphatase in both E. coli DH5alpha and M. smegmatis mc2155, as determined by the formation of blue colonies in the presence of 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine.


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Fig. 1.   Maps of plasmids used in this study.

Glycosylation of the fusion proteins was monitored by their ability to bind ConA following fractionation of recombinant cell extracts by denaturing SDS-PAGE and electrotransfer to nitrocellulose membranes. In these experiments only MPB83-PhoA expressed by M. smegmatis mc2155 was recognized by ConA (Fig. 2A, lane 5). ConA did not bind MPB83-PhoA expressed by E. coli DH5alpha (Fig. 2A, lane 4), demonstrating that post-translational modification of MPB83 is restricted to the mycobacterial host. Neither the MPB70-PhoA fusion protein nor the PhoA protein alone expressed by M. smegmatis mc2155 bound ConA (Fig. 2A, lanes 3 and 6, respectively). Probing a duplicate blot with antibody directed against PhoA (Fig. 2B) demonstrated a similar amount of hybrid protein in each of the extracts, indicating that differences in ConA binding were not a consequence of differences in expression levels of the recombinant fusion proteins. There was evidence of partial proteolysis of fusion proteins expressed in M. smegmatis mc2155 as two bands reacting with anti-PhoA antibody were observed.


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Fig. 2.   Binding of ConA to MPB83 when expressed by mycobacteria. Nitrocellulose membranes incubated with (A) peroxidase-conjugated ConA and (B) rabbit polyclonal antibody raised against bacterial alkaline phosphatase. Lanes: 1, purified PhoA protein; 2, E. coli DH5alpha (p70PhoA); 3, M. smegmatis mc2155 (p70PhoA); 4, E. coli DH5alpha (p83PhoA); 5, M. smegmatis mc2155 (p83PhoA); 6, M. smegmatis mc2155 (19proSP-PhoA).

Immunoblotting with antibodies against PhoA also revealed an increase in the apparent molecular weight of MPB83-PhoA expressed by M. smegmatis mc2155 compared with that of the protein expressed by E. coli DH5alpha (Fig. 2B, lanes 4 and 5). In contrast, no size difference was observed between MPB70-PhoA expressed by M. smegmatis mc2155 or E. coli DH5alpha (Fig. 2B, lanes 2 and 3). The difference in apparent molecular weights of fusion proteins expressed by different bacterial hosts, coupled with the ConA recognition of MPB83-PhoA expressed by M. smegmatis mc2155, supports the conclusion that MPB83 but not MPB70 is glycosylated by mycobacteria.

Identification of a Glycosylation Motif in MPB83 Using PhoA Hybrid Proteins and Site-directed Mutagenesis-- To identify regions of MPB83 involved in ConA binding, in-frame hybrid proteins were produced in which increasing amino-terminal regions of MPB83 were fused to PhoA as described under "Experimental Procedures." This approach was used to generate the set of in-frame fusion proteins listed in Table II.


                              
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Table II
MPB83 amino terminus PhoA fusions

Analysis of ConA binding to these recombinant fusion proteins expressed in M. smegmatis mc2155 demonstrated that proteins containing the first 36 amino acid residues (aa) of MPB83 failed to bind ConA (Fig. 3A, lane 3). However, when the number of amino acids of MPB83 fused to PhoA was increased to 56 or 63, ConA binding was restored (Fig. 3A, lanes 4 and 5). Probing the corresponding blot with antibody to PhoA again demonstrated a similar amount of hybrid protein in each of the extracts (Fig. 3B), indicating that differences in ConA binding were not a consequence of different expression levels of the hybrid proteins. ConA did not bind any of the fusion proteins when expressed by E. coli (data not shown).


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Fig. 3.   Deletion mapping and site-directed mutagenesis of MPB83 to identify regions responsible for ConA binding. Nitrocellulose membranes incubated with peroxidase-conjugated ConA (A and C) and rabbit polyclonal antibody raised against bacterial alkaline phosphatase (B and D). M. smegmatis mc2155 expressing MPB83 amino terminus PhoA fusions from constructs: panels A and B: lanes 1, purified PhoA protein; 2, p28aaPhoA; 3, p35aaPhoA; 4, p56aaPhoA; 5, p63aaPhoA; 6, p83PhoA; and 7, p19proSP-PhoA. Panels C and D: lanes 1, p63aaPhoA; 2, p63aa (TT right-arrow VV)PhoA; 3, p83 PhoA; 4, p19proSP-PhoA.

The amino acid sequence of MPB83 between positions 36 and 56 (37PKPATSPAAPVTTAAMADPA56) contains four potential O-mannosylation sites (Thr41, Ser42, Thr48, and Thr49). Alignment of the amino acid sequences of MPB83 and MPB70 revealed the presence of a cluster of 10 amino acids (residues 46-55) present in MPB83 but absent from MPB70 (highlighted in Fig. 4). This 10-amino acid region showed an overall similarity with the four chemically characterized glycopeptides of MPT32 (16) in having a threonine cluster within a region flanked by proline residues. In light of this similarity the effect of mutagenesis of the two residues (Thr48, Thr49) on ConA binding of p63aaPhoA was investigated. The substitution of threonine to valine was chosen, as the resultant modification, exchange of a methyl group for a hydroxyl group, was the most conservative change with respect to molecular weight.


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Fig. 4.   Comparison of the NH2-terminal amino acid sequence of MPB83 and MPB70. Differences are boxed and the putative O-glycosylation sites are highlighted.

Sonicated extracts of M. smegmatis mc2155 expressing the mutated and native 63aaPhoA fusion protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Incubation of the membrane with peroxidase-labeled ConA revealed that substitution of the Thr48 and Thr49 residues of MPB83 with valine resulted in the complete ablation of ConA binding (Fig. 3C, lane 2). The mutated 63aaPhoA fusion also had a lower apparent molecular weight than that of the native fusion protein (Fig. 3D, lanes 1 and 2). In contrast, the threonine substitutions had little effect on the apparent molecular weight of the fusion construct expressed in E. coli (data not shown).

Purification of MPB83 from M. bovis AN5 Culture Supernatant-- 25- and 23-kDa forms of MPB83 were recognized by the monoclonal antibody MBS43 following the separation of the M. bovis culture supernatant by SDS-PAGE, as shown in Fig. 5B, lane 1. Both molecular weight forms of MPB83 were purified using a chromatography strategy comprising two AEC steps followed by a hydrophobic interaction chromatography step, as described under "Experimental Procedures." The yield of the 23- and 25-kDa forms of MPB83 from a 2-liter culture supernatant were 5 mg and 200 µg, respectively, demonstrating the predominance of the 23-kDa form of MPB83 in the culture supernatant. The purity of the 23-kDa form is shown in Fig. 5. Amino-terminal sequencing data of the 25 kDa showed multiple calling on the NH2-terminal residue with the predominant form shown in Fig. 6, and its low yield is consistent with previous observations that the 25-kDa form is cell-associated (18). It was not possible to make a positive amino acid assignment for the first two residues of the 23-kDa form of MPB83 (Fig. 6); the subsequent residues corresponded to the sequence from Ala50 onwards. Inability to assign amino acids at sites of carbohydrate attachment is a characteristic property of O-glycosylation (33), and it is interesting to note that the unassigned residues correspond to the O-glycosylation sites predicted from the mutagenesis experiments.


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Fig. 5.   Purification of the 23-kDa native form of MPB83 from a M. bovis culture supernatant. A 23-kDa form of MPB83 was purified from a M. bovis AN5 culture supernatant by a 3 column chromatography procedure. The purity of the material following each stage of the purification was determined by silver stain analysis of SDS-PAGE separated samples (panel A). The identity of MPB83 was confirmed by probing the material at each stage in the procedure with a MPB83 specific monoclonal antibody MBS43 (panel B). Lanes 1, M. bovis AN5 culture supernatant (5 µg); 2, purity following DEAE-Fast Flow AEC (2.5 µg); 3, purity following Mono-Q AEC (2.5 µg); 4, purity following hydrophobic interaction chromatography (0.5 µg).


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Fig. 6.   Amino-terminal sequencing of purified forms of MPB83. The predicted amino-terminal sequence of mature MPB83 is shown on line 1. The experimentally determined sequences for the 25- (2) and 23-kDa (3) forms of MPB83 purified from a M. bovis culture supernatant have been aligned against their corresponding positions in the predicted MPB83 sequence.

Definition of MPB83 Glycosylation by Mass Spectrometry-- Nanospray ES-MS of the 23-kDa form of MBP83 on the Q-TOF (28, 29) identified two partially resolved components with masses of 18,048 and 18,066 Da (data not shown). The mass difference of these two components is consistent with partial oxidation of a methionine residue in the sequence to methionine sulfoxide, (a theoretical increase of 16 Da), suggesting that the mass of the 23-kDa form of native MPB83 is 18,048 (± 2) Da. This experimentally calculated mass is 484 (±2) Da greater than that of the theoretical mass of 17,564 calculated from the gene sequence having taken into account the amino acid truncation identified by amino-terminal sequence analysis (Fig. 6). The difference between the experimentally determined and theoretical calculated mass of this form of MPB83 corresponds well with the mass of 3 hexose units (486 Da). In confirmation that MPB83 is modified by the addition of 3 hexose units, chemical deglycosylation of this material by trifluoromethanesulfonic acid produced an unoxidized form of MPB83 with a mass of 17,560 (±2) Da (data not shown). The reduction in mass of MPB83 following the deglycosylation procedure (488 ± 2 Da) confirms the loss of the 3 hexose units. These data demonstrate the effectiveness of the trifluoromethanesulfonic acid deglycosylation procedure in removing glycans while leaving the primary sequence of the protein intact.

To identify the site of glycosylation, a tryptic digest of the 23-kDa form of MPB83 was purified by C18 reverse phase high performance liquid chromatography and fractions were screened for the presence of glycopeptides by ESI-MS on the Q-TOF. The molecule was mapped in its entirety with the exception of TDAK (residues 145-148) and Asp (residue 187). Fraction 36 of the LC trace produced a mass spectrum indicative of the presence of a glycopeptide, with a doubly charged ion at m/z 988. This mass does not map onto the MPB83 molecule, but corresponds to the peptide TTAAMADPAADLIGR (residues 48-62) present as the Met sulfoxide plus 3 hexose units.

The Q-TOF nanospray MS-MS spectrum of the 988 ion is shown in Fig. 7. The spectrum clearly shows the full correct sequence assignment via y" ions (28) at m/z 1388, 1287, 1216, 1145, 998, 927, 812, 715, 644, 573, 458, 345, 232, and 175 plus a number of confirmatory b ions (28), consistent with its identification as a glycosylated derivative of peptide 48-62. Because sugars are lost at relatively low collision energies compared with peptide bond fragmentation, coupled with the poor ion yields at the first position (b1) in a sequence, the spectrum does not allow assignment of which threonine(s) carries the Hex3 mass increment. An experiment was therefore designed to move the first threonine residue of the glycopeptide to a pseudo b2 position by the NH2-terminal reaction with phenylisothiocyanate to form the phenylthiocarbamyl glycopeptide. Q-TOF MS analysis of the phenylisothiocyanate-treated sample confirmed a shift of the m/z 988 signal to a new doubly charged ion at m/z 1056. The MS-MS spectrum of m/z 1056 is shown in Fig. 8. This spectrum is a summation of data acquired at a variety of low collision energies in an attempt to capture peptide fragment ions still retaining the hexose substitutions. A detailed analysis of three of these variable collision energy spectra (Fig. 9), in particular the CAD MS-MS spectrum acquired at 10 eV, reveals the presence of sugar-containing peptide fragment ions corresponding to cleavage between NH2-terminal threonine residues Thr1 and Thr2. These are present as b1 ions carrying the phenylthiocarbamyl group at m/z 237 (Thr1Hex0), 399 (Thr1Hex1), 561 (Thr1Hex2), and 723 (Thr1Hex3), and y14" ions at m/z 1388 (Thr2Hex0), 1550 (Thr2Hex1), 1712 (Thr2Hex2), and 1874 (Thr2Hex3). A confirmatory b13 fragment ion set is also present at m/z 1393, 1555, 1717, and 1879 corresponding to the peptide fragment 48-60 carrying 0-3 hexose residues, respectively. A first-order analysis of peak intensities suggests that a major molecular component of the m/z 1056 [M + 2H]2+ ion is a peptide with Thr1Hex1---Thr2Hex2 glycosylation, although there is certainly evidence from the other signals noted for the presence of less abundant structures in the sample, ranging from Thr1Hex3---Thr2Hex0 through Thr1Hex2---Thr2Hex1 to Thr1Hex0---Thr2Hex3. A further point of interest is that whatever the substitution patterns, only 3 hexose units are attached overall in the principal glycopeptide observed.


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Fig. 7.   ESI (Q-TOF) CAD MS-MS analysis of the [M + 2H]2+ ion at m/z 988. The major fragment ions correspond to the y" series of peptide fragments all of which are lacking sugar attachments (see text).


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Fig. 8.   ESI (Q-TOF) CAD MS-MS analysis of the [M + 2H]2+ ion at m/z 1056, which is the phenylthiocarbamyl derivative of the m/z 988 component. This spectrum is a summation of data obtained over a range of collision energies. For an explanation of the data see discussion of Fig. 9.


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Fig. 9.   ESI (Q-TOF) CAD MS-MS spectra of m/z 1056 obtained at three different collision energies. The spectra are presented in narrow mass ranges for ease of interpretation. Panels A-C, span m/z 150 to 750; panels D-F, span m/z 650 to 1050; and panels G-I, span m/z 1100 to 2200. Panels A, D, and G correspond to 29 eV collision energy, panels B, E, and H correspond to 20 eV collision energy and panels C, F, and I correspond to 10 eV collision energy. For an explanation of signals that were used to assign sugar attachment points and site occupancy see the text.

Identification of the Modifying Sugars and Linkage Analysis-- The identities of the modifying sugars of MPB83 were determined by Dionex ion chromatography following their release by acid hydrolysis as described under "Experimental Procedures." Comparative analysis of the MPB83-hydrolyzed material with standard sugars separated under the same conditions identified a component of MPB83 with an elution time corresponding to mannose (Fig. 10, chromatographs A and B). To confirm that the MPB83 modifying sugar was mannose, the hydrolysis sample was spiked with mannose. Co-elution of the component peaks corresponding to the mannose standard and the sugar detected from MPB83 confirmed the identity of the sugar as mannose (Fig. 10, chromatograph C). Furthermore, the absence of any material with an elution time corresponding to arabinose in the MPB83 sample indicates the absence of sample contamination by the lipopolysaccaride antigen common to many strains of mycobacteria, lipoarabinomannan (LAM). Similar to the observations of Dobos et al. (16) during their characterization of the 45/47-kDa glycoprotein of M. tuberculosis, the minor peaks observed in the MPB83 chromatographs are most likely attributable to components of the chromatographic supports used in its purification.


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Fig. 10.   Analysis of the sugar component of MPB83. MPB83 was subjected to acid hydrolysis and the released sugar present in this material (25 µg) was identified by Dionex ion chromatography (chromatograph B) by comparison with sugar standards run under the same conditions. Chromatograph A demonstrates the elution times of the standard sugars arabinose (0.05 µg), glucose (0.05 µg), and mannose (0.05 µg). Chromatograph C shows the profile of the MPB83 hydrolysis material that was spiked with 0.03 µg of mannose.

The m/z 988 [M + 2H]2+ MPB83 tryptic glycopeptide was reductively eliminated to release the O-glycans, which were permethylated and examined by FAB-MS on a ZAB 2SE 2FPD mass spectrometer. The FAB spectrum (not shown) exhibited a major signal at m/z 493 corresponding to HexHexitol with a minor signal at m/z 697 corresponding to Hex2Hexitol. GC-MS linkage analysis of the partially methylated alditol acetates, derived from the permethylated products of reductive elimination, gave a peak corresponding to terminal mannose (diagnostic fragment ions at m/z 205, 161, 145, 129, and 101) together with a peak that gave a mass spectrum consistent with a linked hexitol. The latter was observed at the elution position corresponding to 3- or 4-linked mannitol. These partially methylated alditol acetates co-elute under the conditions employed in this experiment. Authentic 2-linked mannitol elutes approximately 0.1 min earlier. The fragment ions, which have been shown by examination of authentic standards to be diagnostic of 2-, 3-, and 4-linked hexitols, are shown in Fig. 11A. Significantly, the mass spectrum obtained from the sample hexitol peak was identical to the 3- or 4-mannitol standards and lacked signals at m/z 161 and 129, which are major ions in the spectra of 2-linked hexitols (see Fig. 11A for origin of these ions). This result was surprising because 2-linked mannose is the usual linkage observed in mycobacterial glycopolymers. To confirm our initial observation, and to resolve the ambiguity of 3- and 4-linked mannitol, which give identical mass spectra, the reductive elimination procedure was carried out in duplicate using sodium borodeuteride as the reducing reagent. The deuterium incorporated during the reduction "tags" carbon-1 and therefore allows 3- and 4-mannitol to be differentiated via the shift in mass of the respective fragment ions containing carbon-1. The duplicate experiments gave comparable data and the results from one set of experiments are reproduced. Fig. 11C shows the spectrum of the partially methylated alditol acetates of authentic 3-linked mannitol and Fig. 11B shows the mass spectrum of the peak from the MPB83 sample with the same retention time. All diagnostic fragment ions in the 3-mannitol standard are shared by the sample. Importantly the presence of m/z 206 and the absence of m/z 205 rules out 4-linked mannitol (see Fig. 11A for m/z assignments). Additionally, the absence of m/z 162 and 130, which are the deuterated counterparts of m/z 161 and 129 (see above) and the presence of m/z 90 provides unequivocal evidence that the reducing end mannose is 3-linked and not 2-linked.


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Fig. 11.   Linkage analysis of reductively eliminated glycans from MPB83. A, fragmentation pathways for 2-, 3-, and 4-linked hexitols with and without deuterium at C-1, showing the major fragment ions observed in spectra from authentic standards. B, mass spectrum of the partially methylated alditol acetates of the deutero-reduced hexitol from MPB83, which eluted at the same retention time as authentic deutero-reduced 3-mannitol whose spectrum is shown in C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several of the immunodominant antigens of M. tuberculosis and M. bovis have been reported to be glycosylated (13, 16, 32, 34). However, to date the unambiguous demonstration of mycobacterial glycosylation has been proved only for the M. tuberculosis antigen MPT32 (16). In this paper we have demonstrated that the M. bovis antigen MPB83 is O-glycosylated with mannose via threonine residues. Furthermore, linkage analysis of the carbohydrate reveals a Man(1right-arrow3)Man linkage not previously observed in mycobacterial glycoproteins.

ConA binding activity of mycobacteria-expressed PhoA fusion proteins provides evidence for glycosylation of MPB83 but not of the closely related MPB70 antigen. Further deletion analysis and site-directed mutagenesis identified an essential role in glycosylation for the residues at positions 48 and 49 of MPB83. These residues are not present in the corresponding MPB70 sequence. Direct evidence of glycosylation of Thr48 and Thr49 was obtained by nanospray MS-MS analysis of a tryptic peptide corresponding to MPB83 residues 48-62. The amino acid sequence of the MPB83 glycosylation domain resembles glycosylation sites in MPT32 in the presence of threonine flanked by alanine and proline residues (16).

Whereas there is no clearly defined motif that allows accurate prediction of O-glycosylation sites, the presence of a proline residue -1 or +3 relative to the position of a threonine residue has been found to be useful as a predictor of such sites in eukaryotic mucin-like proteins (35) (www.cbs.dtu.dk/services/ NetOGlyc/). The glycosylation site predicted by site-directed mutagenesis of the 19-kDa antigen of M. tuberculosis is similarly located in a threonine-rich region flanked by proline residues (17). Analysis of MPB83 using this O-glycosylation prediction model identifies nine residues within MPB83 that are potential sites for O-glycosylation. These sites include the threonine doublet at positions 48 and 49. Mass spectrometry analysis demonstrates that this is in fact the only site of glycosylation, at least in the truncated 23-kDa form of MPB83 included in the present study.

In this study Edman degradation amino-terminal sequencing was performed on both the 23- and 25-kDa forms of MPB83. The data shows that the 23-kDa form of MPB83 is generated by proteolytic cleavage immediately before Thr48 from the mature acylated 25-kDa form (18). This raises the possibility that glycosylation may function either as a signal for cleavage or as a means of preventing amino-terminal degradation of the protein following cleavage from its acylated anchor. In support of this latter idea, amino-terminal glycosylation of the cholecystokinin octapeptide (CCK-8) and of glucagon-like peptide-1-(7-36) amide (Tglp-1) leads to their increased resistance to serum aminopeptidase degradation (36, 37). In their study of the M. tuberculosis 19-kDa antigen, Herrmann and colleagues (17) also described a truncated form of the protein in which the glycosylation motif formed the new NH2 terminus. This product was observed when glycosylation had been prevented by threonine to valine substitutions, leading these authors to conclude that glycosylation inhibited the initial proteolytic cleavage from the acylated form of this antigen. This model is not consistent with the findings of this study, where the NH2-terminal threonine of the truncated 23-kDa form is glycosylated. It is interesting to note that such a 23-kDa form is not observed when the gene mpb83 is expressed by M. smegmatis, suggesting that M. bovis may possess peptidases not present in the nonpathogenic mycobacteria.

The dominant glycoform identified for MPB83 was Thr48 substituted with a single mannose residue and Thr49 substituted with a Man(1right-arrow3)Man linkage. However, evidence for a heterogeneous array of glycoforms from Thr48(Man3) Thr49(Man0) through to Thr48(Man0)Thr49(Man3) was also observed. A diversity of mannosylated threonine residues was also seen in the study of the 45/47-kDa antigen of M. tuberculosis, the only other mycobacterial glycoprotein for which the modifying glycans have been fully characterized (16). The exclusive use of mannose in mycobacterial glycoproteins characterized to date differs from the O-linked sugars identified on the glycosylated pilin of N. meningitidis and the glycosylated flagellin of C. jejuni. The predominant modification found in N. meningitidis is a terminal (1 right-arrow 4)-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar that is directly O-linked to the pilin (2), whereas the C. jejuni flagellin was O-glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) (38). The simple O-mannosylation observed for mycobacteria, reminiscent of that seen in yeast, enables us to suggest that the mechanism of mycobacterial glycosylation may also be similar, and may be catalyzed by a family of protein mannosyltransferases as is the case for yeast (39). Protein mannosyltransferase activity has been demonstrated in cell-free extracts from M. smegmatis using peptides from MPT32 and MPB83 as acceptor molecules (40), and experiments in S. coelicolor identify an M. tuberculosis open reading frame, Rv1002c, as a likely candidate for this activity (41).

Although similar to the M. tuberculosis antigen MPT32 in its carbohydrate composition, there is a fundamental difference in the sugar linkages present in the MPB83 glycoprotein. Whereas MPT32 contains (1 right-arrow 2)-linked mannobiose and (1 right-arrow 2),(1 right-arrow 2)-linked mannotriose, the terminal mannose in MPB83 is (1 right-arrow 3)-linked. The Man(1 right-arrow 2)Man linkage seen in MPT32 is also seen as a branching linkage in the mannan backbone of LAM (42), in the di- and trimannosyl units of the Man cap of LAM (43) and as one of the linkages for the mannose present in the phosphatidylinositol mannoside (PIM) family of mycoabacterial phospholipids such as PIM5 and PIM6 (44, 45). In contrast the (1 right-arrow 3) linkage of the mannobiose glycan of MPB83 is not a configuration previously identified within the M. tuberculosis complex. However, a recent report has shown that LAM from the fast growing Mycobacterium chelonae, designated CheLAM, contains a mannan core similar to LAM of M. tuberculosis, but differs in its branching, with LAM having (1 right-arrow 2)-mannose branches as opposed to CheLAM which has (1 right-arrow 3)-mannose branches (46). The genome sequence of M. tuberculosis contains several open reading frames encoding potential mannosyltransferases. Two, pimB and pimC, have been characterized biochemically and shown to encode (1 right-arrow 6)-transferases involved in PIM biosynthesis (47, 48). It will be of interest to identify the (1 right-arrow 3)-transferase, and to determine the factors that influence the preferential (1 right-arrow 2) versus (1 right-arrow 3) modification of the different glycoproteins.

The detailed structural characterization of MPB83 described in this paper provides an essential platform for further exploration of the biological significance of antigen glycosylation by mycobacterial pathogens. Expression of native and site-directed variants of glycoproteins in recombinant hosts will allow assessment of the role of post-translational modification in recognition by the host immune response. In parallel, tools for genetic manipulation of mycobacteria can be used to construct strains deleted for genes encoding individual glycoproteins or enzymes involved in glycosylation, allowing assessment of their contribution to the pathogenesis of tuberculosis.

    ACKNOWLEDGEMENTS

We thank Dr. Jeff Keen for help in NH2-terminal sequencing and Dr. Stephen Gordon for critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by grants from the Department for the Environment Food and Rural Affairs (DEFRA) UK, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust (to A. D. and H. R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§  To whom correspondence should be addressed. Current address: DSTL, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom. Tel.: 44-1980614950; E-mail: slmichell@dstl.gov.uk.

|| Biotechnology and Biological Sciences Research Council Professorial fellow.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M207959200

    ABBREVIATIONS

The abbreviations used are: ConA, concanavalin A; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; AEC, anion exchange chromatography; Q-TOF, orthogonal acceleration time of flight; CAD MS/MS, collisionally activated decomposition tandem mass spectrometry; aa, amino acid(s); LAM, lipoarabinomannan; PIM, phosphatidylinositol mannoside.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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