Genetic Basis for the Synthesis of the Immunomodulatory Mannose Caps of Lipoarabinomannan in Mycobacterium tuberculosis
2006; Elsevier BV; Volume: 281; Issue: 29 Linguagem: Inglês
10.1074/jbc.m603395200
ISSN1083-351X
AutoresPremkumar Dinadayala, Davinder Kaur, Stefan Berg, Anita G. Amin, Varalakshmi Vissa, Delphi Chatterjee, Patrick J. Brennan, Dean C. Crick,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoLipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with α1→2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc2155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell. Lipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with α1→2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc2155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell. Lipoarabinomannan (LAM) 2The abbreviations used are: LAM, lipoarabinomannan; Araf, arabinofuranose; ConA, concanavalin A; GT, glycosyltransferase; GT-C, glycosyltransferase-C; HPAEC, high pH anion exchange chromatography; LM, lipomannan; MALDI-TOF, matrix-assisted laser desorption ionizationtime-of-flight; ManLAM, Manp-capped lipoarabinomannan; Manp, mannopyranose; MTX, 5-deoxy-5-methyl-5-thio-α-xylofuranose; PAS, periodic acid-Schiff; HSQC, heteronuclear single quantum correlation spectroscopy; GC, gas chromatography; MS, mass spectrometry; PI, phosphatidylinositol; MT, mannosyltransferase.2The abbreviations used are: LAM, lipoarabinomannan; Araf, arabinofuranose; ConA, concanavalin A; GT, glycosyltransferase; GT-C, glycosyltransferase-C; HPAEC, high pH anion exchange chromatography; LM, lipomannan; MALDI-TOF, matrix-assisted laser desorption ionizationtime-of-flight; ManLAM, Manp-capped lipoarabinomannan; Manp, mannopyranose; MTX, 5-deoxy-5-methyl-5-thio-α-xylofuranose; PAS, periodic acid-Schiff; HSQC, heteronuclear single quantum correlation spectroscopy; GC, gas chromatography; MS, mass spectrometry; PI, phosphatidylinositol; MT, mannosyltransferase. is a high molecular weight amphipathic lipoglycan, which makes up one of the major components of the cell wall of mycobacteria and exhibits a wide spectrum of immunomodulatory effects. Its structure is complex and heterogeneous with three distinct structural domains, including a phosphatidylinositol anchor (PI anchor), a branched mannan, and a branched arabinan (Fig. 1). The PI anchor is composed of a myo-inositol phosphoryl diacylglycerol substituted at the 2 position with a single mannopyranose (Manp) and at the 6 position with the mannan; this structure is identical to those found in the mycobacterial phosphatidylinositolmannosides and lipomannan (1.Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (113) Google Scholar, 2.Nigou J. Gilleron M. Cahuzac B. Bounery J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), which are thought to be biosynthetic precursors of LAM (3.Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (294) Google Scholar). The mannan core is linked to the 6 position of the myo-inositol residue of the PI anchor and consists of a linear α(1→6) Manp chain with varying degrees of α(1→2) Manp branching with single Manp residues (3.Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (294) Google Scholar). This structure is conserved in all mycobacterial species studied with two exceptions (4.Guerardel Y. Maes E. Elass E. Leroy Y. Timmerman P. Besra G.S. Locht C. Strecker G. Kremer L. J. Biol. Chem. 2002; 277: 30635-30648Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5.Guerardel Y. Maes E. Briken V. Chirat F. Leroy Y. Locht C. Strecker G. Kremer L. J. Biol. Chem. 2003; 278: 36637-36651Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The arabinan component consists of linear stretches of α(1→5) arabinofuranose (Araf) residues with some α(1→3) branching. Two distinct non-reducing termini are generated with a β-d-Araf-(1→2)-α-d-Araf disaccharide linked to the 5 position of α-d-Araf-(1→5)-α-d-Araf-(1→ or the 3 and 5 positions of α-d-Araf-(1→5)-α-d-Araf(1→, resulting in the formation of well characterized Ara4 and Ara6 motifs, respectively, when digested with an endoarabinanase obtained from a Cellulomonas species (Fig. 1; Refs. 3.Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (294) Google Scholar and 6.McNeil M.R. Robuck K.G. Harter M. Brennan P.J. Glycobiology. 1994; 4: 165-173Crossref PubMed Scopus (55) Google Scholar). In slow growing mycobacteria such as Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium avium, and Mycobacterium kansasii (2.Nigou J. Gilleron M. Cahuzac B. Bounery J.D. Herold M. Thurnher M. Puzo G. J. Biol. Chem. 1997; 272: 23094-23103Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 5.Guerardel Y. Maes E. Briken V. Chirat F. Leroy Y. Locht C. Strecker G. Kremer L. J. Biol. Chem. 2003; 278: 36637-36651Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 7.Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1995; 270: 12380-12389Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 8.Khoo H.H. Tang J.B. Chatterjee D. J. Biol. Chem. 2001; 276: 3863-3871Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9.Prinzis S. Chatterjee D. Brennan P.J. J. Gen. Microbiol. 1993; 139: 2649-2658Crossref PubMed Scopus (114) Google Scholar, 10.Chatterjee D. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Glycobiology. 1993; 3: 497-506Crossref PubMed Scopus (77) Google Scholar, 11.Venisse A. Berjeaud J.M. Chaurand P. Gilleron M. Puzo G. J. Biol. Chem. 1993; 268: 12401-12411Abstract Full Text PDF PubMed Google Scholar, 12.Chatterjee D. Lowell K. Rivoire B. McNeil M.R. Brennan P.J. J. Biol. Chem. 1992; 267: 6234-6239Abstract Full Text PDF PubMed Google Scholar) a portion of the non-reducing termini of the Araf chains is capped, to varying degrees, with short α(1→2) Manp chains consisting of one to three residues (Fig. 1), thus the molecule is termed ManLAM. The situation is different in rapidly growing mycobacteria. LAM isolated from Mycobacterium smegmatis and an unidentified Mycobacterium sp. is largely uncapped with a small fraction being capped with inositol phosphate (PILAM) (7.Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 1995; 270: 12380-12389Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13.Gilleron M. Himoudi N. Adam O. Constant P. Venisse A. Riviere M. Puzo G. J. Biol. Chem. 1997; 272: 117-124Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and in Mycobacterium chelonae, no modification exists on the arabinosyl termini (AraLAM) (4.Guerardel Y. Maes E. Elass E. Leroy Y. Timmerman P. Besra G.S. Locht C. Strecker G. Kremer L. J. Biol. Chem. 2002; 277: 30635-30648Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). LAM has been implicated in a plethora of biological functions; typically ManLAM is thought to be anti-inflammatory, while PILAM is thought to be pro-inflammatory. ManLAM has been implicated in inhibition of phagosomal maturation, apoptosis, and interferon-γ signaling in macrophages and interleukin-12 secretion of dendritic cells (reviewed in Refs. 3.Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (294) Google Scholar, 14.Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (216) Google Scholar, and 15.Briken V. Porcelli S.A. Besra G.S. Kremer L. Mol. Microbiol. 2004; 53: 391-403Crossref PubMed Scopus (348) Google Scholar). It has also been suggested that the type of LAM capping is a major structural feature in determining how the immune system is modulated (14.Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (216) Google Scholar), and a recent publication suggests that dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) may act as a pattern recognition receptor and discriminate between Mycobacterium species through selective recognition of the Manp caps on LAM molecules (16.Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. J. Biol. Chem. 2003; 278: 5513-5516Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Thus, LAM structure is generally considered to be a crucial factor in mycobacterial pathogenesis. Despite the level of understanding of the structure of LAM and the variety of immunomodulatory effects it mediates, nothing was known of the enzymes involved in capping of Man-LAM. However, the facts that decaprenylphosphorylarabinofuranose is the only known donor of the Araf residues in the mycobacterial cell wall (17.Wolucka B.A. McNeil M.R. de Hoffmann E. Chojnacki T. Brennan P.J. J. Biol. Chem. 1994; 269: 23328-23335Abstract Full Text PDF PubMed Google Scholar), and synthesis of polar phosphatidylinositolmannosides and linear LM can be inhibited by amphomycin (18.Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19.Morita Y.S. Patterson J.H. Billman-Jacobe H. McConville M.J. Biochem. J. 2004; 378: 589-597Crossref PubMed Google Scholar), suggested that the glycosyltransferases (GTs) involved in the later steps of ManLAM synthesis likely utilize prenylphosphorylglycoses as sugar donors. This idea was supported by the fact that in Corynebacterium glutamicum disruption of polyprenylphosphorylmannose synthase completely obviates lipoglycan synthesis (20.Gibson K.J.C. Eggeling L. Maughan W.N. Krumbach K. Gurcha S.S. Nigou J. Puzo G. Sahm H. Besra G.S. J. Biol. Chem. 2003; 278: 40842-40850Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Therefore, the enzymes involved in ManLAM capping could have structural motifs similar to those reported in GTs that use dolichylphosphorylglycose as sugar donors in eukaryotes (21.Oriol R. Martinez-Duncker I. Chantret I. Mollicone R. Codogno P. Mol. Biol. Evol. 2002; 19: 1451-1463Crossref PubMed Scopus (74) Google Scholar). These eukaryotic GTs have been classified as members of a superfamily of integral membrane GTs (GT-C), that have modified DxD signatures in the first extracellular loop (22.Liu J. Mushegian A. Protein Sci. 2003; 12: 1418-1431Crossref PubMed Scopus (170) Google Scholar). Iterative searches of sequence data bases, motif extraction, structural comparison, and analysis of completely sequenced genomes indicate that members of the GT-C superfamily have limited phylogenetic distribution in that representatives were found in all sequenced eukaryotic genomes, were absent from archaea, and were rare in other prokaryotes with the exception of mycobacteria (22.Liu J. Mushegian A. Protein Sci. 2003; 12: 1418-1431Crossref PubMed Scopus (170) Google Scholar). Approximately 40 putative members of the GT-C superfamily were identified in the genomes of M. tuberculosis H37Rv, M. tuberculosis CDC1551, M. leprae, and M. smegmatis (22.Liu J. Mushegian A. Protein Sci. 2003; 12: 1418-1431Crossref PubMed Scopus (170) Google Scholar). Of these putative GTs 16 were identified in M. tuberculosis H37Rv; these included putative arabinosyltransferases, the mycobacterial Emb proteins, and Rv1002c, which was subsequently shown to catalyze the initial Manp transfer in mycobacterial protein mannosylation (23.VanderVen B.C. Harder J.D. Crick D.C. Belisle J.T. Science. 2005; 309: 941-943PubMed Google Scholar). It was hypothesized that the mannosyltransferases (MTs) involved in capping of ManLAM would be among these 16 putative GTs and would have orthologs in M. leprae, M. avium, and M. bovis but would not have an ortholog in M. smegmatis. Based on this hypothesis MT1671 from M. tuberculosis was identified as a gene encoding a potential GT involved in LAM capping. Therefore, LAM was isolated and structurally characterized from a transposition mutant in which the gene encoding the protein was disrupted and from a recombinant strain of M. smegmatis that expresses the protein. Materials—All chemical reagents were of the highest grade from Sigma unless otherwise specified. M. smegmatis mc2155 was obtained from the American Type Culture Collection. M. tuberculosis CDC1551 (ΔMT1671) strain was obtained by inactivation of the MT1671 gene in a M. tuberculosis ΔSigF background by transposition (24.Lamichhane G. Zignol M. Blades N.J. Geiman D.E. Dougherty A. Grosset J. Broman K.W. Bishai W.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7213-7218Crossref PubMed Scopus (293) Google Scholar, 25.Chen P. Ruiz R.E. Li Q. Silver R.F. Bishai W.R. Infect. Immun. 2000; 68: 5575-5580Crossref PubMed Scopus (118) Google Scholar); these strains along with the parental strain, M. tuberculosis CDC1551, were generously provided by Dr. W. R. Bishai (Center for Tuberculosis Research, The Johns Hopkins University School of Medicine, Baltimore, MD). The insertion of the kanamycin cassette in MT1671 gene was checked by PCR (using primers MT1671f (5′-ATG TTG CTG TGC AAG GCT-3′) and MT1671r (5′-TTA CCG CGT TGA CTT GAC-3′) specific to the MT1671 gene; TSPf (5′-CGC TTC CTC GTG CTT TAC GGT ATC G-3′) and TPSr (5′-CCC GAA AAG TGC CAC CTA AAT TGT AAG CG-3′) specific to the transposon). Culture Conditions—M. smegmatis mc2155 and M. tuberculosis CDC1551 were grown in 7H9 containing oleic acid, albumin, dextrose, and catalase and 0.05% Tween 80. M. smegmatis mc2155-pVV16 and M. smegmatis mc2155-pVV16-MT1671 were grown with kanamycin (50 μg/ml) and hygromycin (50 μg/ml) selection. M. tuberculosis ΔSigF was cultured with hygromycin selection (50 μg/ml), and M. tuberculosis ΔMT1671 was cultured with kanamycin and hygromycin (50 μg/ml each). All bacteria strains were cultured and harvested at late log phase. Construction of Recombinant M. smegmatis mc2155—The 1671-bp open reading frame of MT1671 from M. tuberculosis CDC1551 was amplified from genomic DNA by PCR using primers (MT1671f (5′-TTT TTT CAT ATG CAT GCG AGT CGT CCC G-3′) and MT1671r (5′-TTT TTT AAG CTT ACC GCG TTG ACT TGA CCA C-3′)) engineered to include NdeI and HindIII restriction sites (underlined), respectively. The PCR product was cloned into the vector pGEM (Promega) for sequence confirmation and subsequently ligated into pVV16 derived from pMV261 (26.Stover C.K. de la Cruz V. Fuerst T.R. Burlein J.E. Benson L.A. Bennett L.T. Bansal G.P. Young J.F. Lee M.H. Hatfull G.F. Nature. 1991; 351: 456-460Crossref PubMed Scopus (1182) Google Scholar) after digestion with NdeI and HindIII to create plasmid pVV16-MT1671. M. smegmatis mc2155 was then transformed with pVV16 or pVV16-MT1671 by electroporation. Extraction of LAM and LM—A quick method was used when LAM and LM were extracted from small samples of mycobacteria (50 mg) as described previously (27.Zhang N. Torrelles J.B. McNeil M.R. Escuyer V.E. Khoo K.H. Brennan P.J. Chatterjee D. Mol. Microbiol. 2003; 50: 69-76Crossref PubMed Scopus (109) Google Scholar). Briefly, a mixture of chloroform/methanol/water (10:10:3) was added to the cell pellet and incubated 30 min at 55 °C. The sample was centrifuged, and the organic solvent was removed. Water and phenol saturated with phosphate-buffered saline (1:1) were added to the pellet and then incubated at 80 °C for 2 h. Chloroform was added, and the sample was centrifuged. The supernatant containing LAM and LM was dialyzed against water overnight, and the LAM and LM were analyzed by SDS-PAGE. When LAM and LM were extracted from large quantities of M. tuberculosis for structural analysis the cell pellets from 5-liter cultures were delipidated by serial extractions of 2:1 chloroform/methanol and 10:10:3 chloroform/methanol/water. Subsequently, LAM and LM were extracted essentially as described (8.Khoo H.H. Tang J.B. Chatterjee D. J. Biol. Chem. 2001; 276: 3863-3871Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Wet, delipidated cells were resuspended in breaking buffer (phosphate-buffered saline containing 8% Triton X-114 (Sigma), pepstatin, phenylmethylsulfonyl fluoride, leupeptin, DNase, and RNase). Cells were then disrupted using a French pressure cell. The resulting suspension was centrifuged at 2000 × g for 10 min, and the pellet was discarded. The supernatant was rocked overnight at 4 °C and then centrifuged at 27,000 × g for 15 min. The resulting 27,000 × g pellet was resuspended in breaking buffer and centrifuged as before. The combined supernatant was placed at 37 °C to generate a biphase and centrifuged at 27,000 × g for 15 min at room temperature. The aqueous layer and the detergent layer were back extracted twice, and 9 volumes of cold 95% ethanol was added to the combined detergent layers and incubated at –20 °C overnight. The ethanol precipitate was collected, dried, resuspended in water at 50 mg/ml, digested with 1 mg/ml Pronase (Roche Applied Science), and dialyzed against water. Extraction of LAM and LM from large quantities of M. smegmatis for structural analysis was done as described previously (28.Berg S. Starbuck J. Torrelles J.B. Vissa V.D. Crick D.C. Chatterjee D. Brennan P.J. J. Biol. Chem. 2005; 280: 5651-5663Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Cultures (10 liters) in late log phase were harvested by centrifugation. The cells were delipidated using organic solvents (29.Khoo K.H. Douglas E. Azadi P. Inamine J.M. Besra G.S. Mikusova K. Brennan P.J. Chatterjee D. J. Biol. Chem. 1996; 271: 28682-28690Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and subjected to several freeze-thaw cycles before mechanical disruption by sonication. The resulting suspension was refluxed in 50% ethanol three times. The extracts were pooled and debris removed by centrifugation. The solvent was evaporated, and the sample was resuspended in water at ∼50 mg/ml and digested with 1 mg/ml Proteinase K (Invitrogen). After dialysis the LAM/LM fractions from either M. tuberculosis CDC1551 or M. smegmatis were further purified by size fractionation and analyzed by SDS-PAGE and Western or lectin blotting. Size Fractionation, SDS-PAGE, and Blotting—HPLC size fractionation was performed on a Rainin SD 200 series LC system fitted with a Sephacryl S-200 HiPrep 16/60 column in tandem with a HiPrep 16/60 Sephacryl S-100 column (Amersham Biosciences) at a flow rate of 1 ml/min (29.Khoo K.H. Douglas E. Azadi P. Inamine J.M. Besra G.S. Mikusova K. Brennan P.J. Chatterjee D. J. Biol. Chem. 1996; 271: 28682-28690Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). SDS-PAGE (6% stacking gel and 15% resolving gel) followed by periodic acid-Schiff base (PAS) staining (9.Prinzis S. Chatterjee D. Brennan P.J. J. Gen. Microbiol. 1993; 139: 2649-2658Crossref PubMed Scopus (114) Google Scholar) was used to monitor the elution profile of the fractions containing LAM and LM, which were then pooled and dialyzed. Pooled fractions were re-analyzed by SDS-PAGE to check for purity prior to detailed analysis. Samples were also analyzed by Western blot using monoclonal antibody CS-35 (provided by National Institutes of Health/NIAID contract AI-25469) or lectin blot with concanavalin A (ConA) conjugated to peroxidase (Sigma). LAM and LM were electroeluted from 15% SDS-PAGE to Protran nitrocellulose membranes (Whatman Schleicher and Schuell Bioscience), which were then blocked and incubated with CS-35 (9.Prinzis S. Chatterjee D. Brennan P.J. J. Gen. Microbiol. 1993; 139: 2649-2658Crossref PubMed Scopus (114) Google Scholar) or conjugated ConA. In the case of the Western blots, membranes were incubated with the secondary antibody (anti-mouse IgG coupled to alkaline phosphatase), and color was developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (Sigma). In the case of the lectin blots, compounds were visualized using the 4-chloro-1-naphthol/3,3′-diaminobenzidine, tetrahydrochloride (CN/DAB) substrate kit according to manufacture's instructions (Pierce). Monosaccharide Composition and Linkage Analysis—LAM samples were hydrolyzed with 2 m trifluoroacetic acid, converted to alditol acetates, and analyzed by GC using scyllo-inositol as an internal standard (30.York W.S. Darvill A.G. McNeil M.R. Stevenson T.T. Albersheim P. Methods Enzymol. 1986; 118: 3-40Crossref Scopus (1055) Google Scholar). GC analysis of the alditol acetates was performed on an Hewlett Packard gas chromatograph model 5890 fitted with a SP 2380 column (30 m, 0.25-μm film thickness, 0.25-mm inner diameter; Supelco) using a temperature gradient of 50 °C for 1 min, 30 °C/min to 170 °C, and then 4 °C/min to 260 °C. For linkage analysis, LAM was permethylated using the NaOH/dimethyl sulfoxide slurry method (31.Dell A. Reason A.J. Khoo K.H. Panico M. Mcdowell R.A. Morris H.R. Lennarz W.J. Methods in Enzymology: Guide to Techniques in Glycobiology. Academic Press, Inc., Orlando, FL1994: 108-132Google Scholar), hydrolyzed with 2 m trifluoroacetic acid, and acetylated (30.York W.S. Darvill A.G. McNeil M.R. Stevenson T.T. Albersheim P. Methods Enzymol. 1986; 118: 3-40Crossref Scopus (1055) Google Scholar). GC/MS of the partially methylated alditol acetates was carried out using a ThermoQuest Trace gas chromatograph 2000 (ThermoQuest) connected to a GCQ/Polaris MS mass detector (ThermoQuest). Sample was dissolved in chloroform prior to injection on a DB-5 fused silica capillary column (10 m, 0.18-μm film thickness, and 0.18-mm inner diameter (J&W Scientific)) at an initial temperature of 50 °C, which was held for 1 min. The temperature was then increased to 180 °C over 20 min and then to 250 °C over 8 min. NMR Spectroscopy—Spectra were acquired after several lyophilizations in D2O of 4 mg/0.6 ml in 100% D2O. Two-dimensional 1H-13C heteronuclear single quantum correlation spectroscopy (HSQC) NMR spectra were acquired on a Varian Inova 500-MHz NMR spectrometer using the supplied Varian pulse sequences. The HSQC data were acquired with a 7-kHz window for proton in F2 and a 15-kHz window for carbon in F1. The total recycle time was 1.65 s between transients. Adiabatic decoupling was applied to carbon during proton acquisition. Pulsed field gradients were used throughout for artifact suppression but were not used for coherence selection. The data set consisted of 1000 complex points in t2 by 256 complex points in t1 using States-TPPI (time proportional phase incrementation). Forward linear prediction was used for resolution enhancement to expand t1 to 512 complex points. A cosine-squared weighting function and zero filling were applied to both t1 and t2 prior to the Fourier transformation. The final resolution was 3.5 Hz/point in F2 and 15 Hz/point in F1. Endoarabinanase Digestion and Analysis by HPAEC—LAM (20 μg) was incubated for 16 h at 37 °C with an endoarabinanase isolated from Cellulomonas gelida (3.Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (294) Google Scholar, 6.McNeil M.R. Robuck K.G. Harter M. Brennan P.J. Glycobiology. 1994; 4: 165-173Crossref PubMed Scopus (55) Google Scholar). An aliquot of the digestion mixtures containing both the mannan core and the released oligosaccharides was analyzed directly by Dionex analytical HPAEC performed on a Dionex liquid chromatography system fitted with a Dionex Carbopac PA-1 column. The oligosaccharides were detected with a pulse-amperometric detector (PAD-II) (Dionex). The remaining mixtures were peracetylated as described above and analyzed by MALDI-TOF mass spectrometry. Matrix-assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry—The peracetylated oligosaccharides (10 μg/μl) or the aqueous solutions of the native LAM (10 μg/μl) were mixed with an equal volume of matrix (2,5-dihydroxybenzoic acid dissolved in 10 mg/ml acetonitrile/water, 50:50, 0.1% trifluoroacetic acid) prior to analysis and the molecular mass was measured in negative ion mode by MALDI-TOF on a Bruker Ultraflex TOF/TOF mass spectrometer (Bruker Daltonics). Other Techniques—Protein sequences were obtained from the National Institute for Biotechnology Information (NCBI) world wide web site. BLAST searches were performed at the NCBI site or the TB Structural Genomics Consortium web site. Amino acid sequence alignments were performed using the MultAlin (32.Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4228) Google Scholar) interface on the L'institut National de la Recherché Agronomique Chemin de Borde-Rouge-Auzeville web site. Standard molecular biology techniques were done as described previously (33.Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). Identification of MT1671 as a GT Potentially Involved in LAM Capping—MT1671 from M. tuberculosis CDC1551 met all of the criteria proposed for a GT involved in Manp capping of LAM; it is classified as a putative GT-C GT (22.Liu J. Mushegian A. Protein Sci. 2003; 12: 1418-1431Crossref PubMed Scopus (170) Google Scholar), species of mycobacteria known to have ManLAM have orthologs of this protein, and M. smegmatis, which has PILAM, does not (Fig. 2A). The amino acid sequences of the orthologs in M. tuberculosis H37Rv (Rv1635c) and M. bovis AF2122/97 (Mb1661c) are identical to that of MT1671. M. leprae TN and Mycobacterium avium paratuberculosis also have genes that encode proteins, ML1389 and MAP1338c, respectively, with a high degree of similarity (Fig. 2B). MT1671 is predicted to be a 60.3-kDa protein with an isoelectric point at pH 10.66. The protein is also predicted to have 10 (TMHMM 2.0, PHDhtm) or 11 (SOSUI) transmembrane helices. The program PHDhtm was utilized by Oriol et al. (21.Oriol R. Martinez-Duncker I. Chantret I. Mollicone R. Codogno P. Mol. Biol. Evol. 2002; 19: 1451-1463Crossref PubMed Scopus (74) Google Scholar) to predict the secondary structure of GTs, which use dolichylphosphorylmonosaccharides as the donor substrate, and when used to predict the secondary structure of MT1671, a model was generated in which there were 10 transmembrane helices with the first loop on the extracellular face of the membrane. This loop contains an Asp residue and a Glu residue (DE motif) that are consistent with conserved amino acids in the first, extracellular loop of proteins in the α 2/6 MT superfamily (21.Oriol R. Martinez-Duncker I. Chantret I. Mollicone R. Codogno P. Mol. Biol. Evol. 2002; 19: 1451-1463Crossref PubMed Scopus (74) Google Scholar) and is similar to that predicted for Rv1002c, an enzyme responsible for the addition of a Manp residue to protein acceptors in M. tuberculosis H37Rv (23.VanderVen B.C. Harder J.D. Crick D.C. Belisle J.T. Science. 2005; 309: 941-943PubMed Google Scholar). Characterization of LAM from M. tuberculosis CDC1551 with a Disrupted Copy of MT1671—Since a M. tuberculosis CDC1551 transposon mutant in which MT1671 was disrupted had already been constructed in a ΔSigF background (24.Lamichhane G. Zignol M. Blades N.J. Geiman D.E. Dou
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