Mycobacterium tuberculosis Glycoproteomics Based on ConA-Lectin

Mycobacterium tuberculosis Glycoproteomics Based on ConA-Lectin Affinity Capture of Mannosylated Proteins Margarita Gonza´lez-Zamorano,† Guillermo Mendoza-Hernández,‡ Wendy Xolalpa,† Cristina Parada,† Antonio J. Vallecillo,† Fabiana Bigi,§ and Clara Espitia*,† Departamento de Inmunologıá, Instituto de Investigaciones Biome´dicas, Universidad Nacional Autońoma de Me´xico, C.P. 04510, Me´xico, D.F. Me´xico, Departamento de Bioquı´mica, Facultad de Medicina, Universidad Nacional Autońoma de Me´xico, C.P. 04510, Me´xico, and Instituto de Biotecnologıá, CICVyA-INTA Castelar, Los Reseros y Las Caban ˜ as B1712WAA Hurlingham, Argentina Received September 9, 2008

A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins was resolved by 2-DE gel. After ConA ligand blotting, 41 proteins were identified by mass spectrometry as putative glycoproteins with 34 of them new probably mannosylated proteins. These results contribute to the construction of the ConA affinity glycoprotein database of M. tuberculosis, and provide useful information for understanding the biological role of glycoproteins in mycobacteria. Keywords: M. tuberculosis • mannosylated • ConA affinity • Lipoproteins

Introduction Bacterial genome sequencing and bioinformatics analysis have both played a pivotal role unraveling the glycosylation pathways in prokaryotic organisms. Interestingly, glycans linked to glycoproteins share common biosynthetic precursors in eukaryotes, archaea and prokaryotes, suggesting that glycosylation is a universal modification of proteins that could play similar biological roles in all organisms such as the regulation of structure and function of membrane and secreted proteins.1-3 In recent years, a growing number of bacterial glycoproteins has been identified. Furthermore, the N-glycosylation and O-glycosylation systems have been found in the human gastrointestinal pathogen Campylobacter jejuni.3 N-glycosylation is present in PEB3 and CgpA proteins in which the glycan structure consists of a heptasaccharide of GalNAc with the sugar bacillosamine directly attached to Asn.4 In contrast, O-glycosylation has been found in flagellin, which is modified with monosaccharide analogues to the related sugar pseudaminic acid.5 O-linked flagellar glycosylation has also been described for other pathogenic bacteria including Treponema pallidum, Pseudomonas aeruginosa, Helicobacter pylori, Aeromonas and Clostridium species. Moreover, pilli involved in host-bacteria interactions were found to be O-glycosylated in numerous important pathogens, for example, Neisseria meningitides, Neisseria gonorrheae, P. aeruginosa and Streptococcus parasanguis.6,7 The O-glycosylation pathway in glycoproteins in all these bacteria differs from O-glycosylation of the en* To whom correspondence should be addressed.: Instituto de Investigaciones Biome´dicas Departamento de Inmunologıá., Apartado Postal 70228 04510 D.F. Me´xico D.F., Fax: (5255)56223369, Telephone (5255)56223860, E-mail: [email protected]. † Departamento de Inmunologıá, Instituto de Investigaciones Biome´dicas, Universidad Nacional Autońoma de Me´xico. ‡ Departamento de Bioquı´mica, Facultad de Medicina, Universidad Nacional Autońoma de Me´xico. § Instituto de Biotecnologıá, CICVyA-INTA Castelar. 10.1021/pr800756a CCC: $40.75

 2009 American Chemical Society

doglycosidase glycoprotein of Flavobacterium meningosepticum8 and Apa and Mpb83 glycoproteins of Mycobacterium tuberculosis,9,10 whose glycan structures consist only of mannoses including the sugar covalently linked to the protein. This glycosylation pathway is known as O-mannosylation. It should be noted that, since identifying Apa and PstS1,11 the first M. tuberculosis mannose-containing proteins only mannosylated glycoproteins have been reported in this bacterium.12,13 These proteins were identified by their reactivity with ConA. Analysis of the Apa glycan structure showed four Thr residues glycosylated with R-(1,2)-linked mannose9 in contrast with Mpb83 in which two Thr residues are glycosylated with R-(1,3) linked mannose.10 Apa is also expressed as a recombinant secreted glycoprotein in Streptomyces lividans, and the protein is glycosylated with mannoses in the same amino acid positions as the native protein, providing that the enzymatic machinery for O-mannosylation could be conserved between M. tuberculosis and S. lividans.14 Furthermore, it was revealed that M. tuberculosis membrane-associated Rv1002c, a dolichyl-phosphate-mannose-protein, which requires a lipid carrier to donate mannose, displayed sequence identity with the protein mannosyl transferase (Pmt-1) from Saccharomyces cerevisiae demonstrating conservation of O-mannosylation in M. tuberculosis and eukaryotic organisms.15 In yeast, the impairment of O-mannosylation affects the stability of the cell wall as well as localization, sorting and/or proper function of many proteins that enter the secretory pathway.16,17 Disruption of the pmt gene in Corynebacterium glutamicum was shown to abolish glycosylation of four glycoproteins present in culture supernatants. One of those proteins is the resuscitation promoting factor (Rpf2), which is involved in bacterial growth stimulation and intercellular communication.18 It is important to emphasize that with the exception of Apa, all the glycoproteins in M. tuberculosis found up till now were putative lipoproteins (Lpps). These molecules are a functionally Journal of Proteome Research 2009, 8, 721–733 721 Published on Web 02/06/2009

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Figure 1. ConA ligand blotting assay of M. tuberculosis culture filtrate ConA enriched glycoprotein fractions, CF-EG (A) and CFoEG (B). (A) Lane 1, CF stained with Coomassie blue; lane 2, CFEG stained with Coomassie blue, proteins were eluted with methyl-R-D-mannopyranoside from the ConA column; lane 3, CFEG ligand blotting with ConA; lane 4, CF-EG incubated with ConA in presence of methyl-R-D-mannopyranoside. (B). Lane 1, CFo stained with Coomassie blue: lane 2, CFo-EG stained with Coomassie blue, proteins were eluted with methyl-R-D-mannopyranoside from ConA column; lane 3, CFo-EG ligand blotting with ConA: lane 4, CFo-EG incubated with ConA in presence of methylR-D-mannopyranoside.

diverse class of bacterial proteins characterized by an Nterminal lipid moiety, which serves to anchor the proteins to the cell surface.19 According to estimates based on bioinformatics methods, M. tuberculosis possesses 99 putative Lpps which represent about 2.5% of the predicted M. tuberculosis proteome.20 Herrmann et al. applied NetOglyc analysis to the first 40 N-terminal amino acids of predicted M. tuberculosis Lpps and found that 35 of them possessed sites for Oglycosylation. By expressing the predicted O-glycosylated peptides in the PhoA cassette system, five new mannosylated Lpps were identified based on their reactivity with ConA.13 In this work, we used ConA affinity chromatography combined with bidimensional electrophoresis (2-DE) and ConA ligand blotting to search for mannosylated glycoproteins in M. tuberculosis culture filtrate (CF). The identified spots corresponded to a wide variety of proteins classified in different functional categories. Most of the proteins corresponded to putative Lpps such as LprG and LprA, two antigens that play a role in M. tuberculosis infection and as TLR-2 ligands, induce cytokine responses and regulate antigen presenting cells function.21-25 Finally, it was also interesting to find orthologs of some of the probable mannosylated glycoproteins in the genome of Rhodococcus sp., Nocardia and Streptomyces, diverse actinomycetes closely related to M. tuberculosis. Together these results point to a need of studying the importance of mannosylation in prokaryotic microorganisms and open the possibility of using other bacteria as surrogate hosts to express M. tuberculosis glycoproteins.

Materials and Methods Bacterial Strains and Culture Conditions. The presently used M. tuberculosis H37Rv (ATCC No. 27294) reference strain is kept in the bacterial culture collection at the Instituto de Investigaciones Biome´dicas, UNAM, Mexico City. Bacteria were 722

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Gonza´lez-Zamorano et al. cultured in Proskauer and Beck modified by Youman synthetic medium for 4-6 weeks in static conditions at 37 °C. CF proteins were obtained from the culture medium after elimination of bacilli by filtration. Proteins were precipitated with 0.5 g/mL (NH4)2SO4 at 4 °C and collected by centrifugation at 10 300g for 30 min at 4 °C. The pellet was resuspended in distilled water and dialyzed exhaustively against PBS, pH 7.4. The remaining supernatant of the first precipitation was then saturated with (NH4)2SO4 and proteins were collected under similar conditions to the CF fraction (this fraction was called CF0). Both CF and CF0 proteins were quantified by a modified microtiter plate Lowry assay and stored at -70 °C until needed. ConA-Affinity Chromatography. A 1 mL ConA column (ConA Sepharose Amersham Biosciences) was equilibrated with cationic buffer, pH 6.0 (0.1 M sodium acetate, 1 M NaCl, 1 mM MnCl2 · 4H2O, 1 mM MgCl2 · 6H2O, 1 mM CaCl2). The column was loaded with 1.5 mg of the CF or CF0 fractions, and after several washes with cationic buffer, the proteins were eluted with 0.1 M methyl-R-D-mannopyranoside (Sigma) dissolved in cationic buffer. Fractions of 1 mL were collected and monitored in a spectrophotometer at 280 nm (Jenway 6305). Fractions enriched with putative glycoproteins obtained from CF or CF0 samples were designated as CF-EG and CF0-EG. Samples were lyophilized and stored at -70 °C until needed. Two-Dimensional Polyacrylamide Gel Electrophoresis. The isoelectric focusing was carried out as described elsewhere with minor modifications.26 Briefly, the CF or CF0 and CF-EG or CF0EG fractions were first desalted in a Sephadex G25 column (NAP-5 column, GE Healthcare), then were concentrated by ultrafiltration and requantified. The CF (90 µg of total protein), CF0 (70 µg), CF-EG (70 µg) and CF0-EG (70 µg) fractions were treated with the 2D-Clean Up kit (Amersham Biosciences). The protein pellet was resuspended and the final volume was adjusted to 125 µL with rehydration buffer (8 M urea, 2% CHAPS, 0.5% of IPG buffer pH 4-7, and 20 mM DTT). The sample was used to rehydrate 7 cm immobilized pH 4-7 linear gradient strip (Immobiline DryStrips, GE Healthcare) for 16 h at room temperature (RT), following the manufacturer’s instructions. Focusing started at 300 V (1 h), was increased to 1000 V (0.5 h) and further increased to 5000 V (2 h) in an Ettan IPGphor III Electrophoresis Unit (GE Healthcare). After focusing, the strips were equilibrated for 20 min in equilibrium buffer (2% SDS, 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 0.002% bromophenol blue and 0.5% DTT). The strips were then overlaid onto 12% SDS polyacrylamide gels, and after electrophoresis, proteins were transferred to Immobilon-P PVDF membranes (Millipore, Co.). Membranes were stained with Coomassie Brilliant Blue R-250 and used to perform mass spectrometry (MS), ligand blotting or immunoblotting assays. Ligand Blotting and Immunoblotting. Proteins from the 2-DE and recombinant proteins, LprG and PstS1 (SDS-PAGE resolved) were transferred to PVDF membranes and incubated with 1 µg/mL of ConA-HRP (Sigma) in PBS containing 0.05% Tween 20 and 3% BSA (PBS-TB) in presence or absence of 0.3 M methyl-R-D-mannopyranoside for 1 h at RT. After incubation, membranes were rinsed with PBS containing 0.05% Tween 20 (PBS-T) and ConA reactivity was revealed with 3 mg/mL of 3,3diaminobenzidine in PBS-T and 30% hydrogen peroxide diluted 1:1000. 2-DE membranes containing CF0-EG proteins were also incubated individually for 1 h at RT with rabbit polyclonal antibodies against LprG, PstS1 and mAb IT-19 against LpqH

M. tuberculosis Glycoproteomics

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Figure 2. Ligand blotting assay of M. tuberculosis CF-EG resolved by 2-DE gel. (A) CF-EG proteins blotted onto PVDF membrane and stained with Coomassie blue. (B) Ligand blotting of CF-EG incubated with ConA. (C) CF-EG proteins incubated with ConA in the presence of methyl-R-D-mannopyranoside. (D) Ligand blotting of CF-EG incubated with ConA. Only protein spots that did reacted with ConA consistently over three experiments are circled. The arrows in panel A indicates the position of ConA contaminant spots and the arrow with asterisk shows the nonreacting DnaK spots.

diluted 1:1000 in PBS-TB (mAb IT-19 was donated by TB Research Material and Vaccine Testing Contract, Colorado State University). After incubation, membranes were rinsed twice with PBS-T and incubated for 30 min with protein A-HRP (Zymed) diluted 1:2000 in PBS-TB. After exhaustive rinsing, immunoreactivity was revealed with 3 mg/mL of 3,3-diaminobenzidine in PBS-T and 30% hydrogen peroxide in 1:1000 dilution. SDS-PAGE membranes containing recombinant proteins LprG and PstS1 were immunodetected with the corresponding polyclonal antibodies as described above. Tandem Mass Spectrometry (LC/ESI-MS/MS). The protein spots were carefully excised from the Coomassie stained 2-DE membrane; they were destained, washed, digested with modified porcine trypsin (Promega, Madison, WI) and extracted as previously described.27 The volume of the extracts was reduced by evaporation in a vacuum centrifuge at RT and then adjusted to 20 µL with 1% formic acid.

MS analysis was carried out on a 3200 Q TRAP hybrid tandem mass spectrometer (Applied Biosystems/MDS Sciex, Concord, ON, Canada), equipped with a nano electrospray ion source (NanoSpray II) and a MicroIonSpray II head. The instrument was coupled on-line to a nano Acquity Ultra Performance LC system (Waters Corporations, Milford, MA). Mass calibration of the hybrid triple quadrupole linear ion trap spectrometer was done with polypropylene glycol standard solutions. The instrument was then tuned and tested using [Glu1]-fibrinopeptide B (Sigma). Samples were desalted by injection onto a Symmetry C18 UPLC trapping column (5 µm, 180 µm × 20 mm, Waters Corporations) and washed with 0.1% formic acid in 100% MilliQ water at a flow rate of 15 µL/min. After 3 min, the trap column was switched in-line with the analytical column. Peptides were separated on a BEH, C18 UPLC column (1.7 µm, 75 µm × 100 mm, Waters Corporations) equilibrated with 2% acetonitrile, 0.1% formic acid, using a linear gradient of 2-70% acetonitrile, 0.1% formic acid over a Journal of Proteome Research • Vol. 8, No. 2, 2009 723

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Figure 3. Ligand blotting assay of M. tuberculosis CFo-EG resolved by 2-DE gel. (A) CFo-EG proteins blotted to PVDF membrane and stained with Coomassie blue. (B) Ligand blotting of CFo-EG proteins, incubated with ConA. (C) CFo-EG proteins incubated with ConA in the presence of methyl-R-D-mannopyranoside. (D) CFo-EG membrane incubated with rabbit polyclonal antibody against LprG. The arrows indicate the spots that reacted with the antibody. In panel B, arrows indicate the spots recognized by antibodies against PstS1 and LpqH in a parallel experiment (not shown).

60 min period, at a flow rate of 0.25 µL/min. Spectra were acquired in automated mode using Information Dependent Acquisition (IDA). Precursor ions were selected in Q1 using the enhanced MS mode (EMS) as a survey scan. The scan range for EMS was set at m/z 400-1500 and 4000 amu/s, with an ion spray voltage of +2.2 kV applied to a Picotip emitter FS15020-10-N (New Objective, Woburn, MA). The interface heater for desolvation was held at 150 °C. The survey scan was followed by an enhanced resolution scan (ER) of the three most intense ions at the low speed of 250 amu/sec over a narrow (30 amu) mass range to determine the ion charge states. Two enhanced product ion scans (EPI) of the three most intense peptide signals were performed at 4000 amu/s. The precursor ions were fragmented by collisionally activated dissociation (CAD) in the Q2 collision cell. Collision voltages were automatically adjusted based the ion charge state and mass using rolling collision energy. Generated fragments ions were captured and their masses analyzed in the Q3 linear ion trap. Data interpretation and protein identification were performed with the MS/MS spectra data sets using the MASCOT search algorithm (Version 1.6b9, Matrix Science, London, U.K., available at http://www.matrixscience.com). Searches were conducted using the M. tuberculosis complex subset of the 724


National Center for Biotechnology Information nonredundant database (NCBInr, http://www.ncbi.nih.gov). Trypsin was used as the specific protease, and one missed cleavage was allowed with tolerances of 0.5 Da for the precursor and 0.3 Da for the fragment ion masses. A protein “hit” was accepted as a valid identification when at least one MS/MS spectrum matched at 95% confidence level (p < 0.05). Protein identifications based on a single peptide required a matching probability of >95% and were manually verified. Cloning, Expression and Purification of M. tuberculosis PstS1 and LprG His-Tagged Proteins. The coding region of the pstS1 and lprG genes was amplified by PCR with the high fidelity DNA polymerase Pfx (Invitrogen) from M. tuberculosis H37Rv genomic DNA with the following oligonucleotide primers:pstS1Fo,5′-GGAATTCCATATGGGCTCGAAACCACCGAGCGGT3′ (NdeI site in bold) and pstS1Rv, 5′- CGGGATCCCTAGCTGGAAATCGTCGCGATCAA-3′ (BamHI site in bold), lprgF0, 5′-CCATATGTGCTCGTCGGGCTCGAAG-3′ (NdeI site in bold), lprgRv, 5′- CTGATCAGCTCACCGGGGGCTT-3′ (BclI site in bold). PstS1 (1073 bp) and LprG (889 bp) PCR products were ligated into the pCR4 Blunt-TOPO vector (Invitrogen). The pCR4-lprg construction was replicated in the JM110 Escherichia coli strain (Stratagene) in order to abolish the Dam methylation

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M. tuberculosis Glycoproteomics Table 1. M. tuberculosis CF Enriched-Mannosylated Proteins Identified by LC/ESI-MS/MS spot numbera

1 2 3 4 1-4 1-4 1-4

5 6 7 8 9 6-9 6-9 10 11 12 13 14 15 16 14-16 14-16 14-16 14-16 17 18 18 19 20 21 22 23 24 25 26 22-26 27 28 28 28 29 29 30 30

protein nameb

Not identified Lipoprotein LpqW Lipoprotein LpqW Not identified Lipoprotein LpqW Lipoprotein LpqB Bifunctional membrane-associated penicillin-binding protein 1A/1B PonA2 Not identified Lipoprotein LpqB Lipoprotein LpqB Lipoprotein LpqB Lipoprotein LpqB Lipoprotein LpqB Lipoprotein LpqF Not identified Not identified Secreted protease Secreted protease Not identified Periplasmic oligopeptide-binding lipoprotein OppA Periplasmic oligopeptide-binding lipoprotein OppA Periplasmic oligopeptide-binding lipoprotein OppA Hypothetical protein Rv0907 Gamma-glutamyltranspeptidase precursor GgtB Hypothetical protein Rv0988 Not identified Hypothetical protein Rv0907 Gamma-glutamyltranspeptidase precursor GgtB Hypothetical protein Rv1860 Apa Hypothetical protein Rv1860 Apa Not identified Gamma-glutamyltranspeptidase precursor GgtB Gamma-glutamyltranspeptidase precursor GgtB Gamma-glutamyltranspeptidase precursor GgtB Gamma-glutamyltranspeptidase precursor GgtB Gamma-glutamyltranspeptidase precursor GgtB Gamma-glutamyltranspeptidase precursor GgtB Hypothetical protein Rv1860 Apa Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1 Hypothetical protein Rv2813 Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1 Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1

M. tuberculosis H37Rv gene

NCBI accession number

theoretical M rc

theoretical pIc

Mowse score

matched peptides

sequence coverage %

Rv1166 Rv1166

gi/15608306 gi/15608306

66.1 66.1

5.34 5.34

38 47

5 2

9 3

Rv1166 Rv3244c Rv3682

gi/15608306 gi/15610380 gi/57117145

66.1 61.1 84.5

5.34 5.19 5.34

229 95 66

11 4 5

17 8 7

Rv3244c Rv3244c Rv3244c Rv3244c Rv3244c Rv3593

gi/15610380 gi/15610380 gi/15610380 gi/15610380 gi/15610380 gi/15610729

61.1 61.1 61.1 61.1 61.1 48.4

5.19 5.19 5.19 5.19 5.19 6.49

223 202 216 255 353 27

12 12 11 15 13 1

27 25 23 34 20 2

Rv2672 Rv2672

gi/15609809 gi/15609809

53.9 53.9

4.88 4.88

70 195

1 5

2 10

Rv1280c

gi/15608420

63.4

6.56

51

2

6

Rv1280c

gi/15608420

63.4

6.56

45

1

2

Rv1280c

gi/15608420

63.5

6.56

228

5

12

Rv0907

gi/15608047

56.8

5.43

119

3

7

Rv2394

gi/15609531

66.5

5.66

86

2

4

Rv0988

gi/15608128

42.7

5.26

44

1

2

Rv0907

gi/15608047

56.8

5.43

172

7

13

Rv2394

gi/15609531

66.5

5.66

64

2

3

Rv1860

gi/57116926

32.7

4.52

97

4

34

Rv1860

gi/57116926

32.7

4.52

67

2

12

Rv2394

gi/15609531

66.5

5.66

31

3

7

Rv2394

gi/15609531

66.5

5.66

32

2

3

Rv2394

gi/15609531

66.5

5.66

50

3

7

Rv2394

gi/15609531

66.5

5.66

74

3

5

Rv2394

gi/15609531

66.5

5.66

121

3

5

Rv2394

gi/15609531

66.5

5.66

198

7

10

Rv1860

gi/57116926

32.7

4.52

60

4

33

Rv3006 Rv0934

gi/15610143 gi/57116801

38.7 38.4

4.85 5.14

296 100

16 1

37 4

Rv2813

gi/15609950

29.1

8.6

38

1

1

Rv3006 Rv0934

gi/15610143 gi/57116801

38.7 38.4

4.85 5.14

414 103

16 3

35 10

Rv3006 Rv0934

gi/15610143 gi/57116801

38.7 38.4

4.85 5.14

333 132

12 6

33 24

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Table 1. Continued spot numbera

31 31 31 31 32 32 32 33 33 33 34 34 34 35 35 35 36 36 36 37 38 39 40 41 42 43 43 43 44 44 44 44 45 45 46 46 47 47 47 48

726

protein nameb

Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1 Hypothetical protein Rv1860 Apa Periplasmic phosphate-binding lipoprotein PstS3 Lipoprotein LpqI Glutamine-binding lipoprotein GlnH Lipoprotein LppZ Lipoprotein LpqI Glutamine-binding lipoprotein GlnH Lipoprotein LppZ Glutamine-binding lipoprotein GlnH Lipoprotein LpqI Lipoprotein LppZ Lipoprotein LpqI Lipoprotein LppZ Glutamine-binding lipoprotein GlnH Lipoprotein LpqI Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1 Not identified FEIII-dicitrate-binding periplasmic lipoprotein FecB FEIII-dicitrate-binding periplasmic lipoprotein FecB Lipoprotein LppL Lipoprotein LppL Hypothetical protein Rv1860 Apa Periplasmic phosphate-binding lipoprotein PstS1 Hypothetical protein Rv1860 Apa Periplasmic phosphate-binding lipoprotein PstS2 Periplasmic phosphate-binding lipoprotein PstS1 Periplasmic phosphate-binding lipoprotein PstS2 Lipoprotein LppZ Lipoprotein LpqN Periplasmic phosphate-binding lipoprotein PstS1 Periplasmic phosphate-binding lipoprotein PstS2 Periplasmic phosphate-binding lipoprotein PstS1 Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1 Hypothetical protein Rv0281 Thioredoxin Periplasmic phosphate-binding lipoprotein PstS1



theoretical M rc

theoretical pIc

Mowse score

matched peptides

sequence coverage %

Rv3006 Rv0934

gi/15610143 gi/57116801

38.7 38.4

4.85 5.14

326 243

10 5

26 15

Rv1860

gi/57116926

32.7

4.52

58

1

3

Rv0928

gi/57116798

37.8

5.27

40

3

7

Rv0237 Rv0411c

gi/57116706 gi/15607552

39.3 35.66

5.4 5.39

141 86

5 3

10 8

Rv3006 Rv0237 Rv0411c

gi/15610143 gi/57116706 gi/15607552

38.7 39.3 35.66

4.85 5.4 5.39

48 194 185

2 9 6

7 19 19

Rv3006 Rv0411c

gi/15610143 gi/15607552

38.7 35.66

4.85 5.39

119 411

2 10

8 29

Rv0237 Rv3006 Rv0237 Rv3006 Rv0411c

gi/57116706 gi/15610143 gi/57116706 gi/15610143 gi/15607552

39.3 38.7 39.3 38.7 35.4

5.4 4.85 5.4 4.85 5.39

290 52 172 79 33

10 2 8 3 1

23 6 14 10 2

Rv0237 Rv3006 Rv0934

gi/57116706 gi/15610143 gi/57116801

39.3 38.7 38.4

5.4 4.85 5.14

255 173 40

9 5 2

22 17 9

Rv3044

gi/15610181

36.8

5.2

142

3

8

Rv3044

gi/15610181

36.8

5.2

85

2

5

Rv2138 Rv2138 Rv1860

gi/15609275 gi/15609275 gi/57116926

36.8 36.8 32.7

7.07 7.07 4.52

65 171 28

2 4 1

8 17 6

Rv0934

gi/57116801

38.4

5.14

213

5

18

Rv1860

gi/57116926

32.7

4.52

71

1

3

Rv0932c

gi/57116800

38.1

4.97

58

1

2

Rv0934

gi/57116801

38.4

5.14

347

11

51

Rv0932c

gi/57116800

38.1

4.97

124

2

6

Rv3006 Rv0583c Rv0934

gi/15610143 gi/15607723 gi/57116801

38.7 23.6 38.4

4.85 4.57 5.14

41 31 588

1 1 21

2 4 57

Rv0932c

gi/57116800

38.1

4.97

108

4

13

Rv0934

gi/57116801

38.4

5.14

471

24

53

Rv3006 Rv0934

gi/15610143 gi/57116801

38.7 38.4

4.85 5.02

111 538

2 22

6 50

Rv0281

gi/15607422

33

4.88

53

1

3

Rv1324 Rv0934

gi/15608464 gi/57116801

32.2 38.4

4.63 5.14

34 116

1 7

1 21


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M. tuberculosis Glycoproteomics Table 1. Continued spot numbera

protein nameb



48

Hypothetical protein Rv1084 Peptidyl-prolyl cis-trans isomerase B PpiB Glycosyl hydrolase Peptidyl-prolyl cis-trans isomerase B PpiB Not identified Periplasmic phosphate-binding lipoprotein PstS1 Lipoprotein LpqN Glycosyl hydrolase Periplasmic phosphate-binding lipoprotein PstS1 Glycosyl hydrolase Lipoprotein LpqN Lipoprotein LppZ Periplasmic phosphate-binding lipoprotein PstS1 Glycosyl hydrolase Beta-1,3-glucanase precursor BGlu Beta-1,3-glucanase precursor Bglu Glycosyl hydrolase Beta-1,3-glucanase precursor Bglu Glycosyl hydrolase Lipoprotein LpqN Not identified Cell surface lipoprotein Mpt83 Lipoprotein LpqN Not identified Class A Beta-lactamase BlaC Outer membrane protein A OmpA Periplasmic superoxide dismutase [Cu-Zn] SodC Periplasmic phosphate-binding lipoprotein PstS3 Periplasmic superoxide dismutase [Cu-Zn] SodC Class A Beta-lactamase BlaC Periplasmic superoxide dismutase [Cu-Zn] SodC Lipoprotein LprF Lipoprotein LpqT Lipoprotein LpqT Lipoprotein LprA Cell surface lipoprotein Mpt83 Lipoprotein LprG Lipoprotein LprG Lipoprotein LprG Lipoprotein LprG Lipoprotein LppC Lipoprotein LppX Lipoprotein LppX 19 kDa lipoprotein antigen precursor LpqH 19 kDa lipoprotein antigen precursor LpqH Lipoprotein LppC 19 kDa lipoprotein antigen precursor LpqH Bacterioferritin BfrB Hypothetical protein Rv0020c

Rv1084

gi/15608224

Rv2582

49 49 50 51 52 52 52 53 53 53 53 54 54 54 55 55 56 56 56 57 58 58 59 60 60 60 60 61 61 62 62 63 64 65 65 66 67 68 69 69 70 71 72 73 73 74 74 74

theoretical M rc

theoretical pIc

Mowse score

matched peptides

sequence coverage %

71

5.37

40

4

6

gi/15609719

32.63

9.45

79

1

2

Rv1096 Rv2582

gi/15608236 gi/15609719

31.1 32.6

6.51 9.45

52 64

1 1

3 2

Rv0934

gi/57116801

38.4

5.14

81

1

4

Rv0583c Rv1096 Rv0934

gi/15607723 gi/15608236 gi/57116801

23.7 31.1 38.4

4.57 6.51 5.1

62 51 187

3 1 7

19 3 18

Rv1096 Rv0583c Rv3006 Rv0934

gi/15608236 gi/15607723 gi/15610143 gi/57116801

31.1 23.7 38.7 38.4

6.51 4.57 4.85 5.1

99 89 42 115

3 4 3 1

10 14 5 4

Rv1096 Rv0315

gi/15608236 gi/15607456

31.1 32.2

6.51 4.91

47 44

1 3

3 11

Rv0315

gi/15607456

32.2

4.91

154

7

20

Rv1096 Rv0315

gi/15608236 gi/15607456

31.1 32.2

6.51 4.91

96 134

2 7

6 21

Rv1096 Rv0583c

gi/15608236 gi/15607723

31.1 23.7

6.51 4.57

80 45

1 1

3 4

Rv2873

gi/15610010

22.1

4.86

92

3

18

Rv0583c

gi/15607723

23.6

4.57

45

1

4

Rv2068c

gi/15609205

32.4

5.77

138

4

10

Rv0899

gi/15608039

33.66

6.84

89

1

5

Rv0432

gi/15607573

23.8

5.94

60

1

9

Rv0928

gi/57116798

37.8

5.27

35

1

2

Rv0432

gi/15607573

23.8

5.94

44

4

19

Rv2068c

gi/15609205

32.7

5.77

42

2

4

Rv0432

gi/15607573

23.8

5.94

86

3

18

Rv1368 Rv1016c Rv1016c Rv1270c Rv2873

gi/15608508 gi/15608156 gi/15608156 gi/15608410 gi/15610010

26.9 24.6 24.6 24.8 22.1

8.89 6.51 6.51 5.26 4.86

61 68 70 165 53

1 2 4 13 1

3 8 15 54 8

Rv1411c Rv1411c Rv1411c Rv1411c Rv1911c Rv2945c Rv2945c Rv3763

gi/15608549 gi/15608549 gi/15608549 gi/15608549 gi/15609048 gi/15610082 gi/15610082 gi/15610899

24.5 24.5 24.6 24.6 19.8 24.1 24.1 15.1

7.77 7.77 7.77 7.77 5.76 5.03 5.03 6.54

203 241 159 43 30 58 38 54

8 9 10 2 2 7 1 4

31 31 30 6 7 34 4 47

Rv3763

gi/15610899

15.1

5.76

36

2

28

Rv1911c Rv3763

gi/15609048 gi/15610899

19.8 15.1

5.76 6.54

32 175

1 7

3 47

Rv3841 Rv0020c

gi/15610977 gi/15607162

20.4 56.8

4.73 4.89

58 48

1 2

5 5


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Table 1. Continued spot numbera

protein nameb



theoretical M rc

theoretical pIc

Mowse score

matched peptides

sequence coverage %

74

Hypothetical protein Rv3491 Lipoprotein LppC 19 kDa lipoprotein antigen precursor LpqH 19 kDa lipoprotein antigen precursor LpqH Hypothetical protein Rv2799 Not identified Not identified

Rv3491

gi/15610627

20.3

6.23

40

1

3

Rv1911c Rv3763

gi/15609048 gi/15610899

19.8 15.1

5.76 6.54

34 98

1 2

3 22

Rv3763

gi/15610899

15.1

6.54

63

2

22

Rv2799

gi/15609936

22.8

6.81

41

1

4

74 75 76 76 77 78

a Spot number corresponding to the Figure 2D. b Protein name is based on the data from NCBI database. obtained from Mascot analysis (Matrix Science, London, U.K., available at http://www.matrixscience.com).

Figure 4. Ligand blotting assay of PstS1 and LprG recombinant proteins. (A) CFo-EG. Lane 1, CFo-EG stained with Coomassie blue; lane 2, CFo-EG incubated with ConA; lane 3, CFo-EG incubated with rabbit polyclonal anti-PstS1: lane 4, CFo-EG incubated with rabbit polyclonal anti-LprG. (B) Recombinant PstS1; lane 1, recombinant PstS1, stained with Coomassie blue; lane 2, recombinant PstS1 incubated with ConA; lane 3, recombinant PstS1 incubated with rabbit polyclonal anti-PstS1; lane 4, recombinant PstS1 incubated with monoclonal anti-His antibody. (C) Recombinant LprG; lane 1, recombinant LprG Coomassie blue stained; line 2, recombinant LprG incubated with ConA; lane 3, recombinant LprG incubated with rabbit polyclonal anti-LprG; lane 4, recombinant LprG incubated with monoclonal anti-His antibody.

effect at the BclI restriction site. Captured fragments were subcloned into the pET15b vector, and the identities of the inserts were confirmed by restriction analysis and DNAsequencing. The E. coli strains Rosetta (DE3) (Novagen) and C41 (DE3) (Avidis S.A.) were transformed with pET15b-pstS1 and pET15b-lprG, respectively. Heterologous expression of the PstS1 and LprG proteins was induced in logarithmic phase cultures by adding IPTG (Research Organics) to a final concentration of 0.1 mM. Cells were harvested after 3 h of induction by centrifugation at 4500g for 15 min at 4 °C, and washed with cold PBS. Bacterial pellets were resuspended in PBS (1 g humid weight/3 mL), sonicated and centrifuged at 9000g for 30 min at 4 °C. The PstS-1 inclusion body pellet was washed twice with 2% Triton X-100 in PBS and once only with PBS. The obtained 728


c

Theoretical values of pI and Mr were

inclusion bodies were solubilized in sample buffer (500 mM NaCl, 10 mM imidazol, 8 M urea, 50 mM NaH2PO4,, pH 8.0) with overnight stirring at 4 °C. The suspension was clarified by centrifugation and the supernatant was automatically purified in an AKTA FPLC system (GE Healthcare) using a 1 mL Histrap column (GE Healthcare). Eluted fractions were collected and analyzed by 12% SDS-PAGE. Fractions displaying the recombinant protein were pooled and dialyzed against 0.5 M NaCl, 20 mM NaH2PO4, pH 8.0, with decreasing urea concentrations. LprG recombinant protein was collected in the soluble fraction and purified under the same conditions. Pooled purified fractions were dialyzed against 0.3 M NaCl, 20 mM NaH2PO4, pH 8.0. Both recombinant proteins were quantified by the modified Lowry method and stored at -70 °C until needed. Bioinformatic Analysis. All the sequences identified in the present work were analyzed with software from http://www. expasy.org/tools: LipoP (http://www.cbs.dtu.dk/services/LipoP/) was used to predict Lpps,28 and NetOGgly (http://www.cbs. dtu.dk/services/NetOGlyc/) to predict O-glycosylation.29 SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) to predict signal peptide cleavage sites Protein sequences were downloaded from the NCBI comprehensive microbial resource database (http:// www.ncbi.nlm.nih.gov/genomes/lproks.cgi), For sequence analysis, Blast (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) and Align (http://www.ebi.ac.uk/Tools/emboss/align) were used.

Results ConA-Affinity Chromatography, Mannosylated Glycoproteins Mixture Extracted from CF/CF0 Fractions. CF or CF0 obtained from sequential ammonium sulfate precipitation of culture medium from M. tuberculosis was resolved by 2-DE and their ability to bind to ConA was evaluated by ligand blotting. Several spots showed reactivity with ConA in CF or CF0. Highly specific ConA reactivity was observed with CF proteins within a range of MW 38-45 kDa, but a relatively high background was present at the highest and lowest MW zones. Besides reacting with proteins in the same range as CF, CF0 showed clear and specific ConA reactivity with low molecular weight spots indicating the enrichment of these proteins in the fraction (Supporting Information, Figure S1). It is important to mention that GroEL was detected in the CF by MS illustrating that the 4-6 week-old culture not only contained secreted proteins but also cytoplasmatic proteins. To enrich the potential mannose-containing proteins in CF or CF0,, ConA affinity chromatography was carried out. The CF

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M. tuberculosis Glycoproteomics Table 2. M. tuberculosis ConA Binding Identified Proteins H37Rv gene

protein

spot numbera

signal peptideb

NetOglyc hitsc

Rv0020c Rv0237 Rv0281 Rv0315 Rv0411c Rv0432 Rv0583c Rv0899 Rv0907 Rv0928 Rv0932c Rv0934 Rv0988 Rv1016c Rv1084 Rv1096 Rv1166 Rv1270c Rv1280c Rv1324 Rv1368 Rv1411c Rv1860 Rv1911c Rv2068c Rv2138 Rv2394 Rv2582 Rv2672 Rv2799 Rv2813 Rv2873 Rv2945c Rv3006 Rv3044 Rv3244c Rv3491 Rv3593 Rv3682 Rv3763 Rv3841

Rv0020c LpqI Rv0281 BGlu GlnH SodC LpqN OmpA Rv0907 PstS3 PstS2 PstS1 Rv0988 LpqT Rv1084 Glycosyl hydrolase LpqW LprA OppA Thioredoxin LprF LprG Apa LppC BlaC LppL GgtB PpiB Secreted protease Rv2799 Rv2813 Mpt83 LppX LppZ FecB LpqB Rv3491 LpqF PonA2 LpqH BfrB

74 32-36 47 54-56 32-35 60-62 44, 52, 53, 56, 58 60 14-16, 18 31, 60 43-45 28-31, 36, 43-48, 52-54 14-16 63, 64 48 49, 52-56 1-4, 2, 3 65 14-16 47 62 66-69 19, 20, 27, 31, 42, 43 69, 73, 74 60, 61 40, 41 14-16,18, 22-26 49, 50 12, 13 76 28 58, 65 70, 71 28-36, 44, 46, 53 38, 39 1-4, 6-9 74 6-9 1-4 72-76 74

0.00 1.00 0.07 1.00 0.99 1.00 1.00 0.00 0.00 1.00 1.00 1.00 1.00 0.99 0.00 0.74 1.00 0.99 0.03 0.46 0.99 1.00 1.00 0.97 1.00 1.00 0.99 0.16 0.99 0.57 0.00 1.00 0.99 0.99 0.99 1.00 0.96 1.00 0.98 1.00 0.00

0 12 2 4 10 19 27 8 0 0 2 9 0 20 1 3 27 7 1 4 9 4 14 11 0 5 9 16 6 9 2 9 3 3 3 3 5 2 4 13 1

predicted Lppd

yes

yes yes yes

yes yes yes yes

yes yes yes yes yes yes yes yes yes

yes yes yes yes yes yes yes

proteome reference

30, 31, 54 33, 51 This work 33 33 51 33 This work 51 33, 33, 51, 54, 30-32, 54 This work This work This work This work 33 51 51 31, 51 32 51 30, 31, 54 33 33 This work This work This work 33 33 This work 33 51 33, 51 33 33 33 This work 33 30, 31, 51 33

a Spot number corresponding to Figure 2D. b According to SignalP prediction (http://www.cbs.dtu.dk/services/SignalP/). Using neural hidden Markov models (HMM). c According to NetOglic prediction (http://www.cbs.dtu.dk/services/NetOGlyc/). d According to LipoP algorithm (http://www.cbs.dtu.dk/ services/LipoP/).

or CF0 proteins bound to the ConA-Sepharose column were specifically eluted with methyl-R-D-mannopyranoside. Protein bands in a MW range 21-70 kDa were observed for CF-EG or CF0-EG. Most bands eluted from the column showed reactivity with ConA in a ligand blotting assay and binding was specifically inhibited with methyl-R-D-mannopyranoside (Figure 1). Ligand Blotting Assays of M. tuberculosis CF-EG and CF0-EG Fractions Resolved by 2-DE. M. tuberculosis CF-EG or CF0-EG resolved by 2-DE were evaluated for ConA binding (Figures 2 and 3). Three different experiments were performed and reactive ConA spots were considered positive only if they were represented in the 3 assays. Seventy-eight ConA reactive spots were detected in CF-EG within the range of 15-70 kDa. In some cases, when several positive spots were present at the same molecular weight position, but at low concentrations, they were pooled and only one number was assigned to all the spots (Table 1 and Figure 2D). The presence of the same protein at several spots is a common situation in M. tuberculosis proteomes (http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-

page/extern/index.cgi) that could among other causes be explained by post-translation modification of the proteins. However, specific experimental work would be necessary to define the nature of the different isoelectric forms of the proteins. Coordinates of the major ConA binding spots were matched to protein spots in the Coomassie blue stained membrane and the identity of the spots from CF-EG was revealed by MS (Figure 2A and D). Since comparisons of the CF-EG and CF0-EG 2-DE profiles, based on isoelectric point and molecular mass, and identification of PstS1, Apa and LpqH with antibodies (data not shown) revealed that CF-EG and CF0-EG shared almost the same spots, the latter fraction was not analyzed by MS (Figure 3B). The CF0-EG 2-DE membrane was also incubated with anti-LprG rabbit polyclonal antibody which detects five isoforms of the LprG protein (Figure 3D). In addition, LprG and PstS1 were identified in CF0-EG with rabbit polyclonal antibodies (Figure 4A). The specific interaction of ConA with carbohydrates in Journal of Proteome Research • Vol. 8, No. 2, 2009 729

research articles native proteins was confirmed when PstS1 and LprG, the most abundant proteins in CF0-EG were compared with their respective recombinant proteins for their capacity to interact with ConA. It was clear that non glycosylated E. coli recombinant proteins showed no reactivity with ConA in contrast with the polyclonal antibodies which conserved the capacity to recognize recombinant polypeptides expressed in E. coli (Figure 4B and C). Identification by MS of the ConA Reactive Spots from CF-EG Fraction. The identity of 71 ConA reactive spots in the CF-EG fraction was obtained by MS (Table 1). In some cases, one spot contains more than one protein. Since the proteins were captured by a ConA column, one would expect that most of the spots correspond to glycopeptides, but this still needs to be proved. According to a functional characterization of reported Lpps20 and the TubercuList database (http://genolist.pasteur.fr/TubercuList/), the identified spots include 41 proteins from different functional categories: proteins of unknown function (LpqT, LpqW, LprF, LprG, LppC, LpqB, LppZ, LpqF, LpqN, LppL, Rv0907, Rv0988, Rv1084, Rv2799, Rv2813, and Rv3491); proteins that are predicted to be involved in degradation (glycosyl hydrolases, LpqI and Rv1096, a putative β1-3 glucanase precursor, Rv0315 and a secreted protease, Rv2672); proteins involved in the transport systems of diverse substrates (peptides, a putative glutamine binding lipoprotein, GlnH and OppA, phosphate, PstS1, PstS2 and Psts3 and iron, FecB); enzymes with metabolic and detoxification activities (γglutamyl transferase GgtB, superoxide dismutase SodC, a transpeptidase PonA2, a putative S-adenosylmethionine-dependent methyltransferase Rv0281, a thioredoxin, Rv1324 a peptidylprolyl isomerase PpiB); proteins with a role in adhesion and cell invasion (Apa, LpqH, Mpt83), a putative porin, OmpA a possible bacterioferritin, BfrB and a presumed beta lactamase BlaC; proteins with alleged roles in signaling and its related functions (LprA, LppX, LprG). It is important to note that 11 of the spots identified by MS, OmpA, LprF, GgtB, LpqF, LppL, Rv0281, Rv0988, Rv1084, Rv1096, Rv1234 and Rv2582, corresponded to proteins that have not been previously reported in M. tuberculosis proteomic publications,30-33 nor in the Statens Serum Institut or in the Max Planck Institute for Infection Biology proteomic databases (http://www.ssi.dk/sw14644.asp, http://web.mpiib-berlin. mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi) (Table 2). We also found that among spots that did not bind to ConA in the ligand blotting assay is the heat-shock protein DnaK. On the other hand, most non-ConA reacted spots corresponded to ConA itself (Figure 2A). In this regard, a complete inhibition of ConA binding was achieved in the presence of methyl-R-Dmannopyranoside (Figure 2C). Bioinformatics Analysis. All proteins identified by MS in the present work were analyzed for the presence of a potential signal peptide and O-glycosylation sites. As shown in Table 2, most of them possess a potential site for type I signal peptidase and show 1-27 potential O-glycosylation sites. The sequence of each of the identified proteins was obtained and Blast protein analysis was carried out. Lpps showed a significant percentage of identity with potential lipoglycoproteins from Rhodococcus sp. RHA1 (Supporting Information, Table 1). In accordance with these findings was the high identity (61.6%) and similarity (75.4%) between the O-mannosyltranferase of Rhodococcus sp. RHA1_ro05660 and Rv1002. 730


Gonza´lez-Zamorano et al. The evolutionary relationship between the actinomycetes enzymes is illustrated in Supporting Information Figure S2.

Discussion ConA lectin affinity chromatography combined with 2-DE and MS was used in this work to search for new mannosylated glycoproteins in the M. tuberculosis proteome. Our results demonstrated that M. tuberculosis possesses a high number of putative mannosylated glycoproteins with a wide range of different functions. Such a variety of functions linked to mannosylation suggests that this could be a common posttranslational modification for proteins exported to the cell wall or extracellular environment. Protein glycosylation coupled with the secretory pathway has been established in the yeast S. cerevisiae.1 Moreover, it was recently shown that specific translocation processes were required for protein O-mannosylation in mycobacteria.15 O-mannosylation is a very important post-translational modification in yeast, which determines the structure and integrity of the cell walls as well as cellular differentiation and virulence.7,16,17 In mammals and insects, Pmt proteins are essential for cellular differentiation and development, while lack of Pmt activity causes Walker-Warburg syndrome (muscular dystrophy) in humans.7 It is relevant that the majority of the putative glycoproteins identified in the present work corresponded to Lpps, a group of mycobacterial molecules involved in virulence as demonstrated by the attenuation in virulence of the M. tuberculosis lpsA mutant. The lpsA gene encoded a signal peptidase involved in Lpp processing.34 Some of the proteins that have been classified as glycoproteins are potent antigens like PstS1, LpqH, Mpt83 and Apa.35-37 Although their biological functions have not been well-defined yet, it is known that the PstS1 and Mpt83 genes are part of operons, PstS1 of the phosphate transport operon, and mpt83 is cotranscribed with mpt70.38-40 The role of the glycans in O-mannosylated proteins is unknown; however, mannose linked to the proteins could be important for the immune response. Native purified Apa induced a potent delayed-type hypersensitivity response and was shown to stimulate primed T-cells in vitro. In contrast, the nonglycosylated recombinant protein expressed in E. coli did not induce a delayed-type hypersensitivity or T-cell stimulatory response.41,42 Likewise, antibodies from tuberculosis patients recognized the glycosylated protein but not the nonglycosylated form.14 Other important features of mycobacterial glycolipoproteins have been related to their capacity to interact with the host through their glycan structures. It has been recently demonstrated that LpqH is one of the major adhesins for macrophage mannose receptor and DC-SIGN;43,44 Apa also binds to DC-SIGN and surfactant protein, interactions that could facilitate colonization and invasion of host cells.44,45 Both LpqH and PstS1 interact with Toll-like receptors; however while the interaction of LpqH with TLR-2 inhibits IFNγ_regulated MHC-II expression in alveolar macrophages,37 PstS1 acting through both TLR-2 and TLR-4 induces the activation of pathways, which in turn play an essential role in TNF-R and IL-6 expression during mycobacterial infection.46 Finally, among the Lpps identified in this work was LprG.21 This protein is considered a virulence factor since a knockout of the M. tuberculosis lprG gene has proved to be attenuated in virulence in a mouse tuberculosis model.47 LprG abrogated the protection afforded by the BCG vaccine, suggesting that the Lpp may modulate the course of experimental infection and could play a role in tuberculosis infection by inducing increased suppres-


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22,48

putative M. tuberculosis mannosylated proteins were found, most of them Lpps. Since Lpps are involved in the immune response to M. tuberculosis as well as in virulence, it is tempting to think that the mannosylated Lpps found in this work could play similar roles.

sion of the immune response. The LprG antiprotective effect could be due to the capacity of LprG to modulate the immune response through activation of TLR-2, an activity also found in LprA.25 Interestingly, LprG also requires glycosylation for MHC class II-restricted T cell activation in vivo.23,24 Regarding the biological role of this lipoprotein, it has been recently reported that LprG acts in cooperation with P55, the major facilitator superfamily small molecule transporter of ethidium bromide, suggesting that the protein is necessary for P55 mediated-transport across the cell membrane.49,50 Taken together, these results suggest that many of the new putative glycoproteins identified in the present work could represent potential adhesins for DC-SIGN and mannose receptors, and they could also be important proteins involved in virulence and immune response to mycobacteria. It is important to note that the strategy of enrichment glycoprotein with ConA allowed us to identify new proteins unreported in M. tuberculosis proteome databases. Because the analyzed proteins were obtained by ConA chromatography and their reactivity with ConA was confirmed by ligand blotting, it is reasonable to think that most glycoproteins identified in this work contain mannose. In addition, for most, the glycosylation sites were predicted with NetOglyc and it is still possible that some nonpredicted proteins could be true glycoproteins since the prediction method is not 100% accurate; it has been mainly used to predict glycosilation sites in mammalian proteins but it is also used to predict mannose O-glycosylated sites in fungi (http://www.cbs.dtu.dk/services/NetOGlyc-2.0/abstract.php). Whether or not they are O-mannosylated proteins will need to be tested experimentally. It is strongly expected to find the Lpps associated with cell membranes19,20,51 and we have found an unexpected number of putative lipoglycoproteins in our 4-6 week old static cultures. Their presence in CF could be due to bacterial lysis; however, some Lpps have been found consistently in culture supernatants, even in short-term cultures31,52 Another possible explanation could be that they are being proteolytically released from their N-terminal anchors perhaps by a type I signal peptidase. Interesting predicted sites for the enzyme were observed for many of the Lpps found in this work. Furthermore, the conservation of the O-mannosylation pathway between M. tuberculosis and S. cerevisiae suggests that O-mannosylation could be the only form of O-glycosylation present in mycobacteria that also occurs in Saccharomyces.1,15 Surprisingly, many of the mycobacterial Lpps previously described20 and those found in the present work have orthologues in other actinomycetes. The high degree of homology found with Lpps from Rhodococcus sp. RHA1 was significant. This bacterium is a soil microorganism which catabolizes a wide range of compounds and represents a genus of considerable industrial interest.53 The homology of Rhodococcus sp. with putative M. tuberculosis lipoglycoproteins and with the Omannosyl transferase suggest that the O-mannosylation machinery is strongly conserved between the two organisms and Rhodococcus sp. could offer advantages such as being used as a surrogate expression host to produce M. tuberculosis recombinant glycoproteins and also to study glycosylation in mycobacteria.

Conclusion A profile of putative mannosylated proteins from a M. tuberculosis culture filtrate enriched in mannose containing proteins was resolved by 2-DE gel. A high number of new

Acknowledgment. We thank Erika Segura for technical assistance and Isabel Perez Montfort who corrected the English version of this manuscript. G.M.-H. thanks Waters (Me´xico) for providing the nanoUPLC for this work. This work was supported by grants from CONACyT (G36923-M, 33580-M), DGPA (IN221599) Universidad Nacional Autonoma de Me´xico. Supporting Information Available: Figure S1, ligand blotting assay of M. tuberculosis CF resolved by 2-DE. (A) CF proteins blotted onto PVDF membrane stained with Coomassie blue; (B) ligand blotting of CF incubated with ConA; (C) CF proteins incubated with ConA in the presence of methyl-R-Dmannopyranoside; (D) CFo proteins blotted onto PVDF membrane stained with Coomassie blue; (E) CFo incubated with ConA; (F) CFo proteins incubated with ConA in the presence of methyl-R-D-mannopyranoside. Figure S2, unrooted phylogenetic tree for dolichyl-phosphate-mannose-protein mannosyltransferase Rv1002c and related proteins. The values are relative evolutionary distance. The tree was generated using a ClustalW 2.0 alignment and CLC Main Workbench 4.1.2. Proteins are from M. tuberculosis (Rv1002c); Rhodococcus sp. RHA1 (RHA1_ro05660); Mycobacterium marinum (MMAR_4491); Mycobacterium avium 104 (MAV_1126); Mycobacterium ulcerans Agy99 (MUL_4663); Mycobacterium leprae (ML0192); Mycobacterium smegmatis MC2 155 (MSMEG_5447); Nocardia farcinica IFM 10152. (Nfa49110); Corynebacterium glutamicum (Cg1014); Kineococcus radiotolerans SRS30216 (Krad_3667); Arthrobacter aurescens TC1 (AAur_1329); Streptomyces coelicolor A3 (SCO3154); Janibacter sp. HTCC2649 (JNB_16958); Renibacterium salmoninarum 33209 (RSAL 33209_3002); Clavibacter michiganensis (CMS_2336); Leifsonia xyli (Lxx17570); Brevibacterium linens (BlinB01001696); Actinomyces odontolyticus (ACTODO_02212); (Bifidobacterium dentium) (BIFDEN_00955); (S. cerevisiae (PMT1); Homo sapiens (POMT1). Table S1, actinomycetes proteins related to M. tuberculosis H37Rv putative glycoproteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Herscovics, A.; Orlean, P. Glycoprotein biosynthesis in yeast. FASEB J. 1993, 7 (6), 540–550. (2) Benz, I.; Schmidt, M. A. Never say never again: protein glycosylation in pathogenic bacteria. Mol. Microbiol. 2002, 45 (2), 267– 276. (3) Szymanski, C. M.; Logan, S. M.; Linton, D.; Wren, B. W. Campylobacter a tale of two protein glycosylation Systems. Trends Microbiol. 2003, 11 (5), 233–238. (4) Linton, D.; Dorrell, N.; Hitchen, P. G.; Amber, S.; Karlyshev, A. V.; Morris, H. R.; Dell, A.; Valvano, M. A.; Aebi, M.; Wren, B. W. Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway. Mol. Microbiol. 2005, 55 (6), 1695–1703. (5) Thibault, P.; Logan, S.; M; Kelly, J. F.; Brisson, J. R.; Ewing, C. P.; Trust, T. J.; Guerry, P. Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin. J. Biol. Chem. 2001, 276 (37), 34862–34870. (6) Schmidt, M. A.; Riley, L. W.; Benz, I. Sweet new world: glycoproteins in bacterial pathogens. Trends Microbiol. 2003, 11 (12), 554– 561. (7) Lengeler, K. B.; Tielker, D.; Ernst, J. F. Protein-O-mannosyltransferases in virulence and development. Cell. Mol. Life. Sci. 2008, 65 (4), 528–544.


research articles (8) Plummer, T. H., Jr.; Tarentino, A. L.; Hauer, C. R. Novel, specific O-glycosylation of secreted Flavobacterium meningosepticum proteins. Asp-Ser and Asp-Thr-Thr consensus sites. J. Biol. Chem. 1995, 270 (22), 13192–13196. (9) Dobos, K. M.; Khoo, H.-K.; Swiderek, K. M.; Brennan, P. J.; Belisle, J. T. Definition of the full extent of Glycosylation of the 45kilodalton glycoprotein of Mycobacterium tuberculosis. J. Bacteriol. 1996, 178 (9), 2498–2506. (10) Michell, S. L.; Whelan, A. O.; Wheeler, P. R.; Panico, M.; Easton, R. L.; Etienne, A. T.; Haslam, S. M.; Dell, A.; Morris, H. R.; Reason, A. J.; Herrmann, J. L.; Young, D. B.; Hewinson, R. G. The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1 f 3)-mannobiose moieties. J. Biol. Chem. 2003, 278 (18), 16423– 16432. (11) Espitia, C.; Mancilla, R. Identification, isolation and partial characterization of Mycobacterium tuberculosis glycoprotein antigens. Clin. Exp. Inmmunol. 1989, 77 (3), 378–383. (12) Garbe, T.; Harris, D.; Vordermeier, M.; Lathigra, R.; Ivany, J.; Young, D. Expression of the Mycobacterium tuberculosis 19 kDa Antigen in Mycobacterium smegmatis: Immunological analysis and evidence of glycosylation. Infect. Immun. 1993, 61 (1), 260–267. (13) Herrmann, J. L.; Delahay, R.; Gllagher, A.; Robertson, B.; Young, D. Ana´lisis of post-translational modification of mycobacterial proteins using a cassette expresioń system. FEBS Lett. 2000, 473 (3), 358–362. (14) Lara, M.; Servıń-Gonza´lez, L.; Singh, M.; Moreno, C.; Cohen, I.; Nimtz, M.; Espitia, C. Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans. Appl. Environ. Microbiol. 2004, 70 (2), 679–685. (15) VanderVen, B. C.; Harder, J. D.; Crick, D. C.; Belisle, J. T. Exportmediated Assembly of mycobacterial glycoproteins parallels eucaryotic pathways. Science. 2005, 309 (5736), 941–943. (16) Proszynski, T. J.; Simons, K.; Bagnat, M. O-glycosylation as a sorting determinant for cell surface delivery in yeast. Mol. Biol. Cell. 2004, 15 (2), 1533–1543. (17) Willer, T.; Brandl, M.; Sipiczki, M.; Strahl, S. Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast. Mol. Microbiol. 2005, 57 (1), 156–170. (18) Mahne, M.; Tauch, A.; Pu ¨ hler, A.; Kalinowski, J. The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins. FEMS Microbiol. Lett. 2006, 259 (2), 226– 233. (19) Rezwan, M.; Grau, T.; Tschumi, A.; Sander, P. Lipoprotein synthesis in mycobacteria. Microbiology. 2007, 153 (3), 652–658. (20) Sutcliffe, I. C.; Harrington, D. J. Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components. FEMS Microbial Rev. 2004, 28 (5), 645–659. (21) Bigi, F.; Espitia, C.; Alito, A.; Zumarraga, M.; Romano, M. I.; Cravero, S.; Cataldi, A. A novel 27 kDa lipoprotein antigen from Mycobacterium bovis. Microbiology 1997, 143 (Pt11), 3599–3605. (22) Hovav, A-H.; Mullerad, J.; Davidovitch, L.; Fishman, Y.; Bigi, F.; Cataldi, A.; Cercovier, H. The Mycobacterium tuberculosis recombinant 27-kilodalton lipoprotein induces a strong Th1-type immune response deleterious to protection. Infect. Immun. 2003, 71 (6), 3146–3154. (23) Gehring, A. J.; Dobos, K. M.; Belisle, J. T.; Harding, C. V.; Boom, H. W. Mycobacterium tuberculosis LprG (Rv1411c): A novel TLR-2 ligand that inhibits macorphage class II MHC antigen processing. J. Immunol. 2004, 173 (4), 2660–2668. (24) Sieling, P. A.; Hill, P. J.; Dobos, K. M.; Brookman, K.; Kuhlman, A. M.; Fabri, M.; Krutzik, S. R.; Rea, T. H.; Heaslip, D. G.; Belisle, J. T.; Modlin, R. L. Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation. J. Immunol. 2008, 180 (9), 5833–5842. (25) Pecora, N. D.; Gehring, A. J.; Canaday, D. H.; Boom, W. H.; Harding, C. V. Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function. J. Immunol. 2006, 177 (1), 422–429. (26) Xolalpa, W.; Vallecillo, A. J.; Lara, M.; Mendoza-Hernańdez, G.; Comini, M.; Spallek, R.; Singh, M.; Espitia, C. Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis. Proteomics 2007, 7 (18), 3332–3341. (27) Bienvenut, W. V.; Sanchez, J. C.; Karmime, A.; Rougue, V.; Rose, K.; Binz, P. A.; Hochstrasser, D. F. Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot. Anal. Chem. 1999, 71 (21), 4800–4807.

732


Gonza´lez-Zamorano et al. (28) Juncker, A. S.; Willenbrock, H.; Von Heinje, G.; Nielsen, H.; Brunak, S.; Krogh, A. Prediction of lipoprotein signal peptides in Gramnegative bacteria. Protein Sci. 2003, 12 (8), 1652–1662. (29) Julenius, A.; Mølgaard, R.; Gupta, K.; Brunak, S. Prediction, conservation analysis and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 2005, 15 (2), 153–164. (30) Rosenkrands, I.; Weldingh, K.; Jacobsen, S.; Hansen, C. V.; Florio, W.; Gianetri, I.; Andersen, P. Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 2000, 21 (5), 935–948. (31) Rosenkrands, I.; King, A.; Weldingh, K.; Moniatte, M.; Moertz, E.; Andersen, P. Towards the proteome of Mycobacterium tuberculosis. Electrophoresis 2000, 21 (17), 3740–3756. (32) Sinha, S.; Arora, S.; Kosalai, K.; Namane, A.; Pym, A.; Cole, S. T. Proteome analysis of the plasma membrane of Mycobacterium tuberculosis. Comp. Funct. Genomics 2002, 3 (6), 470–483. (33) Målen, H.; Berven, F. S.; Fladmark, K. E.; Wiker, H. G. Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv. Proteomics 2007, 7 (10), 1702–1718. (34) Sander, P.; Rezwan, M.; Walker, B.; Rampini, S. K.; Kroppenstedt, R. M.; Ehlers, S. C.; Keller, J. R.; Keeble, M.; Hagemeier, M. J.; Colston, M. J.; Springer, B.; Böttger, E. C. Lipoprotein processing is required for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 2004, 52 (6), 1543–1552. (35) Espitia, C.; Cervera, I.; Gonza´lez, R.; Mancilla, R. A 38-kD Mycobacterium tuberculosis antigen associated with infection. Its isolation and serologic evaluation. Clin. Exp. Immunol. 1989, 77 (3), 373–377. (36) Hewinson, R. G.; Michell, S. L.; Russell, W. P.; McAdam, R. A., Jr. Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70. Immunol. 1996, 43 (5), 490–499. (37) Noss, E. H.; Pai, R. K.; Sellati, T. J.; Radolf, J. D.; Belisle, J.; Golenbock, D. T.; Boom, W. H.; Harding, C. V. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19 kD lipoprotein of Mycobacterium tuberculosis. J. Immunol. 2001, 167 (5), 910–918. (38) Braibant, M.; Lefe´vre, P.; De Wit, L.; Peirs, P.; Ooms, J.; Huygen, K.; Andersen, A. B.; Content, J. A Mycobacterium tuberculosis gene cluster encoding protein of a phosphate transporter homologous to the Escherichia coli Pst system. Gene 1996, 176 (1-2), 171–176. (39) Torres, A.; Jua´rez, M. D.; Cervantes, R.; Espitia, C. Molecular analysis of Mycobacterium tuberculosis phosphate specific transport system in Mycobacterium smegmatis. Characterization of recombinant 38 kDa (PstS-1). Microb. Pathog. 2001, 30 (5), 289– 297. (40) Jua´rez, M. D.; Torres, A.; Espitia, C. Characterization of the Mycobacterium tuberculosis regioń containing the mpt83 and mpt70 genes. FEMS Microbiol. Lett. 2001, 203 (1), 95–102. (41) Horn, C.; Namane, A.; Pescher, P.; Rivie`re, M.; Romain, F.; Puzo, G.; Baˆrzu, O.; Marchal, G. Decreased capacity of recombinant 45/ 47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern. J. Biol. Chem. 1999, 274 (45), 32023–32030. (42) Romain, F.; Horn, C.; Pescher, P.; Namane, A.; Riviere, M.; Puzo, G.; Barzu, O.; Marchal, G. Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses. Infect. Immun. 1999, 67 (11), 5567–5572. (43) Silvestre, H.; Espinosa, P.; Sańchez, A.; Esparza, M.; Pereira, A. L.; Bernal, G.; Espitia, C.; Mancilla, R. The 19 kDa antigen of Mycobacterium tuberculosis is a major adhesine that binds the mannose recptor of TPH-1 monocytic cells and promotes phagocytosis of mycobacteria. Microb. Pathog. 2005, 39 (3), 97–107. (44) Pitarque, S.; Herrmann, J. L.; Duteyrat, J. l.; Jackson, M.; Stewart, G. R.; Lecointe, F.; Payre, B.; Schwartz, O.; Young, B. D.; Marchal, G.; Lagrange, P. H.; Puzo, G.; Gicquel, B.; Nigou, J.; Neyrolles, O. Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity. J. Biochem. 2005, 392 (Pt3), 615–624. (45) Ragas, A.; Roussel, L.; Puzo, G.; Rivie´re, M. The Mycobacterium tuberculosis Cell-surface glycoprotein Apa as a potencial adhesin to colonize target cells via the innate immune system Pulmonary C-type Lectin surfactante Protein A. J. Biol. Chem. 2007, 282 (8), 5133–5142. (46) Jung, S. B.; Yang, C. S.; Lee, J. S.; Shin, A. R.; Jung, S. S.; Son, J. W.; Harding, C. V.; Kim, H. J.; Park, J. K.; Paik, T. H.; Song, C. H.; Jo, E. K. The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release

research articles


(47)

(48)

(49)

(50) (51)

of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes. Infect. Immun. 2006, 74 (5), 2686–2696. Bigi, F.; Gioffre´, A.; Klepp, L.; Santangelo, M. P.; Alito, A.; Caimi, K.; Meikle, V.; Zuma´rraga, M.; Taboga, O.; Rommano, M. I.; Cataldi, A. The knockout of the lprG-Rv1410 operon produces strong attenuation of Mycobacterium tuberculosis. Microbes Infect. 2004, 6 (2), 182–187. Hovav, A.; Mullerad, J.; Maly, A.; Davidovitch, L.; Fishman, Y.; Bercovier, H. Aggravated infection in mice co-administered with Mycobacterium tuberculosis and the 27-kDa lipoprotein. Microbes Infect. 2006, 8 (7), 1750–1757. Silva, P. E. A.; Bigi, F.; Santangelo, M. de la P.; Romano, M. I.; Martın, C.; Cataldi, A.; Aıńsa, J. A. Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2001, 45 (3), 800–804. Farrow, M. F.; Rubin, E. J. Function of a mycobacterial major facilitator superfamily pump requires a membrane-associated lipoprotein. J. Bacteriol. 2008, 190 (5), 1783–1791. Mattow, J.; Siejak, F.; Hagens, K,; Schmidt, F.; Koehler, C-; Treumann, A.; Schaibl, U. E.; Kaufmann, S. H. Improved strategy

for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria. Proteomics 2007, 7 (10), 1687–1701. (52) Pheiffer, C.; Betts, J. C.; Flynn, H. R.; Lukey, P. T.; van Helden, P. Protein expression by a Beijing strain differs from that of another clinical isolate and Mycobacterium tuberculosis H37Rv. Microbiology 2005, 151 (Pt 4), 1139–1150. (53) McLeod, M. P.; Warren, R. L.; Hsiao, W. W.; Araki, N.; Myhre, M.; Fernandes, C.; Miyazawa, D.; Wong, W.; Lillquist, A. L.; Wang, D.; Dosanjh, M.; Hara, H.; Petrescu, A.; Morin, R. D.; Yang, G.; Stott, J. M.; Schein, J. E.; Shin, H.; Smailus, D.; Siddiqui, A. S.; Marra, M. A.; Jones, S. J.; Holt, R.; Brinkman, F. S.; Miyauchi, K.; Fukuda, M.; Davies, J. E.; Mohn, W. W.; Eltis, L. D. The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl. Acad. Sci. U.S.A. 2006, 103 (42), 15582– 15587. (54) Max Planck Institute Protein Database: http://web. mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/menu_frame.cgi?gel).

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