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• W2067539471 abstract "The dimannoside (PIM2) and hexamannoside (PIM6) phosphatidyl-myo-inositol mannosides are the two most abundant classes of PIM found in Mycobacterium bovis bacillus Calmette Guérin, Mycobacterium tuberculosis H37Rv, and Mycobacterium smegmatis 607. Recently, these long known molecules received a renewed interest due to the fact that PIM2 constitute the anchor motif of an important constituent of the mycobacterial cell wall, the lipoarabinomannans (LAM), and that both LAM (phosphoinositol-capped LAM) and PIM are agonists of Toll-like receptor 2 (TLR2), a pattern recognition receptor involved in innate immunity. Due to the biological importance of these molecules, the chemical structure of PIM was revisited. The structure of PIM2 was recently published (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., Gachelin, G., and Puzo, G. (2001) J. Biol. Chem. 276, 34896–34904). Here we report the purification and molecular characterization of PIM6 in their native form. For the first time, four acyl forms of this molecule have been purified, using hydrophobic interaction chromatography. Mono- to tetra-acylated molecules were identified in M. bovis bacillus Calmette Guérin, M. tuberculosis H37Rv, and M. smegmatis 607 using a sophisticated combination of analytical tools, including matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and two-dimensional homo- and heteronuclear NMR spectroscopy. These experiments revealed that the major acyl forms are similar to the ones described for PIM2. Finally, we show that PIM6, like PIM2, activate primary macrophages to secrete TNF-α through TLR2, irrespective of their acylation pattern, and that they signal through the adaptor MyD88. The dimannoside (PIM2) and hexamannoside (PIM6) phosphatidyl-myo-inositol mannosides are the two most abundant classes of PIM found in Mycobacterium bovis bacillus Calmette Guérin, Mycobacterium tuberculosis H37Rv, and Mycobacterium smegmatis 607. Recently, these long known molecules received a renewed interest due to the fact that PIM2 constitute the anchor motif of an important constituent of the mycobacterial cell wall, the lipoarabinomannans (LAM), and that both LAM (phosphoinositol-capped LAM) and PIM are agonists of Toll-like receptor 2 (TLR2), a pattern recognition receptor involved in innate immunity. Due to the biological importance of these molecules, the chemical structure of PIM was revisited. The structure of PIM2 was recently published (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., Gachelin, G., and Puzo, G. (2001) J. Biol. Chem. 276, 34896–34904). Here we report the purification and molecular characterization of PIM6 in their native form. For the first time, four acyl forms of this molecule have been purified, using hydrophobic interaction chromatography. Mono- to tetra-acylated molecules were identified in M. bovis bacillus Calmette Guérin, M. tuberculosis H37Rv, and M. smegmatis 607 using a sophisticated combination of analytical tools, including matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and two-dimensional homo- and heteronuclear NMR spectroscopy. These experiments revealed that the major acyl forms are similar to the ones described for PIM2. Finally, we show that PIM6, like PIM2, activate primary macrophages to secrete TNF-α through TLR2, irrespective of their acylation pattern, and that they signal through the adaptor MyD88. A variety of phosphatidyl-myo-inositol mannosides (PIM) 1The abbreviations used are: PIM, phosphatidyl-myo-inositol mannosides; BCG, bacillus Calmette Guérin; BLP, bacterial lipopeptide; C16, palmitate; C18, stearate; C19, tuberculostearate (10-methyloctadecanoate); COSY, correlation spectroscopy; ESI-MS, electrospray ionization-mass spectrometry; Gro, glycerol; HABA, 2-(4-hydroxyphenylazo)-benzoic acid; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; LAM, lipoarabinomannans; LM, lipomannans; ManLAM, LAM with mannosyl extensions; MALDI-Tof-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry; myo-Ins, myo-inositol; MyD88, myeloid differentiation factor; p, pyranosyl; PI, phosphatidyl-myo-inositol; QMA, quaternary methyl ammonium; ROESY, rotating frame nuclear Overhauser effect spectroscopy; t, terminal; TLR, Toll-like receptor; TMS, trimethylsilyl; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide.-based compounds are known to be part of the mycobacterial cell wall. Among these compounds are the lipoarabinomannans (LAM) and the lipomannans (LM). The importance of these lipoglycans in the immunopathogenesis of tuberculosis is now established, and a rising number of studies in the literature are devoted to the delineation of their biological activities (2Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar, 3Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (219) Google Scholar). PIM, LM, and LAM derive from the same biosynthetic pathway as demonstrated using biochemical (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 2Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar, 4Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (116) Google Scholar, 5Besra 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 (146) Google Scholar) and genetic approaches (6Schaeffer M.L. Khoo K.H. Besra G.S. Chatterjee D. Brennan P.J. Belisle J.T. Inamine J.M. J. Biol. Chem. 1999; 274: 31625-31631Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 7Kremer L. Gurcha S.S. Bifani P. Hitchen P.G. Baulard A. Morris H.R. Dell A. Brennan P.J. Besra G.S. Biochem. J. 2002; 363: 437-447Crossref PubMed Google Scholar, 8Kordulakova J. Gilleron M. Mikusova K. Puzo G. Brennan P.J. Gicquel B. Jackson M. J. Biol. Chem. 2002; 277: 31335-31344Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). PIM appear to be the common anchor of LM and LAM, as LM correspond to polymannosylated PIM and then give rise to LAM by further glycosylation with arabinosyl units. This anchor plays a fundamental role in the biological functions of LAM. Indeed, it is now clearly established that most of the LAM immunoregulatory effects are abolished by alkaline hydrolysis, highlighting the importance of the lipidic part of the anchor (2Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar). Mycobacterium bovis BCG 1173P2 (the Pasteur strain) (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), Mycobacterium smegmatis ATCC-607 (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and Mycobacterium tuberculosis H37Rv ATCC-27294 were found to mainly contain two PIM families, the dimmanosylated (PIM2) and the hexamannosylated (PIM6) ones. PIM1, PIM3, PIM4, and PIM5 were observed in very small amounts, suggesting that they are biosynthetic intermediates. PIM are known from the 1940s and have been structurally investigated by Ballou and co-workers in the 1960s (9Lee Y.C. Ballou C.E. Biochemistry. 1965; 4: 1395-1404Crossref PubMed Scopus (85) Google Scholar). By 1965, studies of deacylated PIM from M. tuberculosis and Mycobacterium phlei revealed the structure of the saccharidic part. PIM6 were the highest PIM which were fully characterized from M. phlei (10Hill D.L. Ballou C.E. J. Biol. Chem. 1966; 241: 895-902Abstract Full Text PDF PubMed Google Scholar) and were shown to contain a pentamannoside of sequence Manpα1→2Manpα1→2Manpα1→6Manpα1→6Manpα1→attached to position 6 of the myo-inositol, whereas a Manp unit is linked to the position 2 of the myo-inositol. Recently, the complete structure of native PIM2 has been achieved (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 2Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar). These last studies focused on the characterization of their lipidic part and unambiguously established the existence of a tetra-acylated form that was thus far suggested (4Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (116) Google Scholar). Several biological functions have been recently attributed to PIM. PIM2 were shown to recruit natural killer T cells, which have a primary role in the local granulomatous response (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11Apostolou I. Takahama Y. Belmant C. Kawano T. Huerre M. Marchal G. Cui J. Taniguchi M. Nakauchi H. Fournie J.J. Kourilsky P. Gachelin G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5141-5146Crossref PubMed Scopus (182) Google Scholar). Moreover, a role for surface-exposed PIM as M. tuberculosis adhesins that mediate attachment to non-phagocytic cells has also been established (12Cywes C. Hoppe H.C. Daffe M. Ehlers M.R. Infect. Immun. 1997; 65: 4258-4266Crossref PubMed Google Scholar, 13Hoppe H.C. de Wet B.J. Cywes C. Daffe M. Ehlers M.R. Infect. Immun. 1997; 65: 3896-3905Crossref PubMed Google Scholar). Analysis of infected macrophages revealed that PIM, among other mycobacterial lipids, are actively trafficked out of the mycobacterial phagosome (14Beatty W.L. Rhoades E.R. Ullrich H.J. Chatterjee D. Heuser J.E. Russell D.G. Traffic. 2000; 1: 235-247Crossref PubMed Scopus (297) Google Scholar). This could be of particular importance relating to the potential role played by these constituents in extending the influence of the bacterium over its surroundings. An unfractionated preparation of PIM, as well as phosphoinositol capped LAM (PILAM), was recently shown to activate cells via Toll-like receptor-2 (TLR-2) (15Jones B.W. Means T.K. Heldwein K.A. Keen M.A. Hill P.J. Belisle J.T. Fenton M.J. J. Leukoc. Biol. 2001; 69: 1036-1044PubMed Google Scholar). Activation of TLR-dependent signaling pathways leads to the activation of genes that participate in innate immune responses, such as expression of cytokines, coactivation molecules, and nitric oxide (16Heldwein K.A. Fenton M.J. Microbes Infect. 2002; 4: 937-944Crossref PubMed Scopus (134) Google Scholar, 17Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4767) Google Scholar). Finally, PIM6 as well as ManLAM from Mycobacterium leprae and M. tuberculosis are presented by antigen-presenting cells in the context of CD1b (18Sieling P.A. Chatterjee D. Porcelli S.A. Prigozy T.I. Mazzaccaro R.J. Soriano T. Bloom B.R. Brenner M.B. Kronenberg M. Brennan P.J. Modlin R.L. Science. 1995; 269: 227-230Crossref PubMed Scopus (675) Google Scholar). The high affinity interaction of CD1b molecules with the PIM2 acyl side chains was then established (19Ernst W.A. Maher J. Cho S. Niazi K.R. Chatterjee D. Moody D.B. Besra G.S. Watanabe Y. Jensen P.E. Porcelli S.A. Kronenberg M. Modlin R.L. Immunity. 1998; 8: 331-340Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The phosphatidylinositol moiety plays a central role in the process of PIM and ManLAM binding to CD1b proteins. Here we investigated the structure of the most polar PIM isolated from M. bovis BCG, PIM6. The earlier NMR studies conducted on PIM2 and PIM6 by Severn et al. (20Severn W.B. Furneaux R.H. Falshaw R. Atkinson P.H. Carbohydr. Res. 1998; 308: 397-408Crossref PubMed Scopus (27) Google Scholar) focused on the deacylated molecules, thus excluding the study of the lipidic moieties. In this study, we investigated the chemical structure of native PIM6, focusing on the characterization of the different “acyl forms,” using sophisticated analytical tools such as MALDI-MS and two-dimensional NMR. Then we demonstrated the capacity of the PIM2 and PIM6 acyl forms to stimulate macrophages to produce cytokines, and we investigated the implication of the different TLR in this process. PIM Extraction—The PIM-containing lipidic extract was obtained through purification of the phenolic glycolipids from M. bovis BCG 1173P2 (the Pasteur strain) (21Vercellone A. Puzo G. J. Biol. Chem. 1989; 264: 7447-7454Abstract Full Text PDF PubMed Google Scholar) and was briefly summarized in Ref. 1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar. An acetone-insoluble phospholipids-containing lipid extract was prepared (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and applied to a QMA-Spherosil M (BioSepra SA, Villeneuvela-Garenne, France) column that was first irrigated with chloroform, chloroform/methanol (1:1, v/v), methanol in order to elute neutral compounds. Phospholipids were eluted using ammonium acetate containing organic solvents. Indeed, 0.1 m ammonium acetate in chloroform/methanol (1:2, v/v) (fraction A) allowed elution of 750 mg of phospholipids (enriched in phosphatidyl-myo-inositol di-mannosides (PIM2)),and 0.2 m ammonium acetate in chloroform/methanol (1:2, v/v) (fraction B) was subdivided into two fractions. The first volumes allowed elution of 440 mg of phospholipids (cardiolipids essentially), and the next ones allowed elution of 160 mg of phospholipids (mixture of phosphatidyl-myo-inositol dimannosides (PIM2) and hexamannosides (PIM6)), and finally, 0.2 m ammonium acetate in methanol (fraction C) allowed elution of 55 mg of phospholipids (enriched in PIM6). Repeated lyophilizations were necessary to eliminate ammonium acetate salts. PIM from M. smegmatis (ATCC 607) and M. tuberculosis H37Rv (ATCC 27294) were also analyzed. 2M. Gilleron, V. F. J. Quesniaux, and G. Puzo, unpublished data. Purification of the PIM Acyl Forms—Fraction C (20 mg) was loaded in 0.1 m ammonium acetate solution containing 15% (v/v) propanol-1 to octyl-Sepharose CL-4B (Amersham Biosciences) column (20 × 1.5 cm) pre-equilibrated with the same buffer. The column was first eluted with 50 ml of equilibration buffer and then with a linear propanol-1 gradient from 15 to 65% (v/v) (250 ml each) in 0.1 m ammonium acetate solution at a flow rate of 5 ml/h. The fractions were collected every 30 min. 20 μl of each fraction was dried and submitted to acidic hydrolysis (100 μl of trifluoroacetic acid, 2 m, 2 h at 110 °C). The hydrolysates were dried, reconstituted in water, and then analyzed by high pH anion exchange chromatography for mannose content giving the presented chromatogram (Fig. 2A). Fractions were pooled according to the purification profile, and repeated lyophilizations were performed to eliminate ammonium acetate salts. An acetone precipitation step was done on each fraction in order to eliminate contaminants issued from the propanol-1. Finally, 1.2 (fraction I), 1 (fraction II), 7.5 (fraction III), and 3 mg (fraction IV) were obtained. Purification was checked by TLC on aluminum-backed plates of silica gel (Alugram Sil G, Macherey-Nagel, Duren, Germany), using chloroform/methanol/water, 60:35:8 (v/v/v), as migration solvent. A sulfuric anthron spray and a Dittmer-Lester spray were used to detect carbohydrates containing lipids and phosphorus-containing lipids, respectively. Acetolysis Procedure—200 μg of PIM were treated with 200 μl of anhydrous acetic acid-d 4/acetic anhydride-d 6, 1:1 (v/v), at 110 °C for 12 h. The reaction mixture was dried under stream of nitrogen and was submitted to acetylation. The mixture was dissolved in acetic anhydride/anhydrous pyridine, 1:1 (v/v), at 80 °C for 2 h. The reaction mixture was dried under stream of nitrogen. 20 μl of chloroform/methanol, 9:1 (v/v), was added and analyzed in MALDI-Tof-MS in positive and negative modes. Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-Tof-MS)—Analysis by MALDI-Tof-MS was carried out on a Voyager DE-STR (PerSeptive Biosystems, Framingham, MA) using the reflectron mode. Ionization was effected by irradiation with pulsed UV light (337 nm) from an N2 laser. PIM were analyzed by the instrument operating at 20 kV in the negative ion mode using an extraction delay time set at 200 ns. Typically, spectra from 100 to 250 laser shots were summed to obtain the final spectrum. All of the samples were prepared for MALDI analysis using the on-probe sample cleanup procedure with cation-exchange resin (22Ludwiczak P. Brando T. Monsarrat B. Puzo G. Anal. Chem. 2001; 73: 2323-2330Crossref PubMed Scopus (47) Google Scholar). The HABA matrix (from Sigma) was used at a concentration of ∼10 mg/ml in ethanol/water (1:1, v/v). Typically, 0.5 μl of PIM sample (10 μg) in a CHCl3/CH3OH/H2O solution and 0.5 μl of the matrix solution, containing ∼5–10 cation exchange beads, were deposited on the target, mixed with a micropipet, and dried under a gentle stream of warm air. The measurements were externally calibrated at two points with PIM. NMR Analysis—NMR spectra were recorded with an Avance DMX500 spectrometer (Bruker GmbH, Karlsruhe, Germany) equipped with an Origin 200 SGI using Xwinnmr 2.6. Samples were dissolved in CDCl3/CD3OD/D2O, 60:35:8 (v/v/v), and analyzed in 200 × 5-mm 535-PP NMR tubes at 308 K. Proton chemical shifts are expressed in ppm downfield from the signal of the chloroform (δH/TMS 7.27 and δC/TMS 77.7). The one-dimensional phosphorus (31P) spectra were measured at 202 MHz with phosphoric acid (85%) as external standard (δp 0.0). All the details concerning NMR sequences used and experimental procedures were detailed in previous study on PIM2 (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Primary Macrophage Cultures—TLR2- and/or TLR4-deficient mice obtained by inter-cross from TLR4-deficient mice (23Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar) and TLR2-deficient mice (24Michelsen K.S. Aicher A. Mohaupt M. Hartung T. Dimmeler S. Kirschning C.J. Schumann R.R. J. Biol. Chem. 2001; 276: 25680-25686Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), TLR6-deficient mice (25Takeuchi O. Kawai T. Muhlradt P.F. Morr M. Radolf J.D. Zychlinsky A. Takeda K. Akira S. Int. Immunol. 2001; 13: 933-940Crossref PubMed Scopus (1010) Google Scholar) and MyD88-deficient mice (26Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar), and their control littermates were bred under specific-pathogen-free conditions in the Transgenose Institute animal breeding facility (Orléans, France). Murine bone marrow cells were isolated from femurs and cultivated (106/ml) for 7 days in Dulbecco's minimal essential medium supplemented with 20% horse serum and 30% L929 cell-conditioned medium (as source of M-CSF, as described in Ref. 27Muller M. Eugster H.P. Le Hir M. Shakhov A. Di Padova F. Maurer C. Quesniaux V.F. Ryffel B. Mol Med. 1996; 2: 247-255Crossref PubMed Google Scholar). Three days after washing and re-culturing in fresh medium, the cell preparation contained a homogenous population of macrophages. The bone marrow-derived macrophages were plated in 96-well microculture plates (at 105 cells/well) and stimulated with LPS (Escherichia coli, serotype O111:B4, Sigma, at 100 ng/ml), bacterial lipopeptide (Pam3-Cys-Ser-Lys4, EMC microcollections; at 0.5 μg/ml), or PIM preparations at the indicated concentration. Lyophilized PIM preparations were solubilized in Me2SO and added in the cultures at a non-cytotoxic final concentration of 1–1.5% Me2SO (cell viability monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Alternatively the macrophages were infected with M. bovis BCG (Pasteur strain 1173P2; kind gift from G. Marchal, Pasteur Institute, Paris, France; at a multiplicity of infection of 2 bacteria per cell). After 18 h of stimulation, the supernatants were harvested and analyzed for cytokine content using commercially available enzyme-linked immunosorbent assay reagents for TNF-α and IL-12p40 (Duoset R & D Systems, Abingdon, UK). Purification of the PIM 6 Acyl Forms from M. bovis BCG—An enriched fraction of phosphatidyl-myo-inositol hexamannosides (PIM6) was purified from M. bovis BCG as described previously (fraction C (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar)). Briefly, PIM are known to be found in the acetone-insoluble fraction of mycobacterial lipidic extract (28Gilleron M. Vercauteren J. Puzo G. J. Biol. Chem. 1993; 268: 3168-3179Abstract Full Text PDF PubMed Google Scholar). The contaminating neutral compounds were eliminated by QMA anion exchange chromatography, irrigated with neutral eluents. The phospholipids were then eluted with ammonium acetate-containing organic solvents resulting in three fractions, A–C. These fractions were analyzed by ESI-MS in negative mode (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and revealed that fraction A mainly includes PIM2 containing a total of three and four fatty acids in addition to phosphatidyl-myo-inositol; fraction B contains PIM2 and PIM6 containing a total of three and four fatty acids, and fraction C mainly contains the different acyl forms of PIM6. Fig. 1 presents the negative MALDI spectrum of M. bovis BCG fraction C that is dominated by peaks assigned to deprotonated molecular ions (M – H)– revealing the different acyl forms of PIM6 present in this fraction. From the predominant fatty acids deduced from gas chromatography-mass spectrometry analysis (not shown), i.e. palmitic (C16), tuberculostearic (C19), and stearic (C18) acids and in minor amounts heptadecanoic acids (C17), the fatty acid composition of each acyl form was determined. We then confirmed mono-acylated forms (1Ac) with C16 (m/z 1543.6) or C19 (m/z 1585.7), di-acylated forms (2Ac) with 2C16 (m/z 1781.8) or 1C16,1C19 (m/z 1823.9), tri-acylated forms (3Ac) mainly with 2C16,1C19 (m/z 2062.1), and tetra-acylated forms (4Ac) mainly constituted by 3C16,1C19 (m/z 2300.3) or 2C16,2C19 (m/z 2342.4). M. tuberculosis H37Rv was found to produce as major PIM families PIM2 and PIM6 in the same acyl forms as the ones described for M. bovis BCG (not shown). To proceed in the separation of the acyl forms, 20 mg of fraction C were applied on an octyl-Sepharose column, using propanol-1 as eluent (Fig. 2A). The different acyl forms were eluted at concentrations of propanol-1 ranging from 25 to 50% and separated into 4 sub-fractions according to the profile elution determined by the mannose content. Each sub-fraction (I to IV) was collected and analyzed by negative MALDI-Tof-MS. MALDI-Tof-MS Characterization of the Octyl-Sepharose Column Fractions—The mass spectra of the fraction I (Fig. 2, B–C) revealed that it contained mono-acylated forms of the molecules (1Ac). Indeed, the analysis of fraction Ia revealed deprotonated molecular ions at m/z 1543.6 characterizing mono-acylated forms with C16 (Fig. 2B), whereas mono-acylated forms with C19 appeared more abundant in fraction Ib (m/z 1585.7) (Fig. 2C). The mass spectrum corresponding to fraction II showed three peaks with an intensity above 10% and were assigned to (M – H)– ions of di-acylated forms: two major peaks at m/z 1781.8 and 1823.9 and one minor one at m/z 1795.8 (Fig. 2D). The two major peaks were characterized as (M – H)– ions of di-acylated forms containing 2C16 and 1C16,1C19, respectively, and the minor peak corresponded to (M – H)– ions of the molecules acylated by 1C16,1C17. The mass spectrum of fraction III (Fig. 2E) revealed two peaks of intensity superior to 10% assigned to tri-acylated forms of the molecules. The major peak at m/z 2062.1 was attributed to (M – H)– ions corresponding to tri-acylated forms containing 2C16,1C19, whereas the minor one at m/z 2090.1 contains 1C16,1C18, and 1C19. The mass spectrum of fraction IV appeared more complex, constituted by a series of peaks (Fig. 2F) between m/z 2230.2 and 2356.4 and assigned to (M – H)– ions of different tetra-acylated forms. Indeed, the most abundant (M – H)– ions at m/z 2300.3 and 2342.4 characterized tetra-acylated forms containing 3C16,1C19 and 2C16,2C19, whereas the ions at m/z 2314.3 corresponded to tetra-acylated forms containing 2C16,2C18. Minor ions at m/z 2272.3, 2286.3, 2328.4, and 2356.4 were attributed to the molecules esterified by 3C16,1C17, 3C16,1C18, 2C16,1C18,1C19, and 1C16,2C18,1C19 or 1C16 and 1C17,2C19, respectively. In addition, species esterified by unsaturated fatty acids were also present as each peak consisted of a –2 analog. Therefore, we have developed a powerful preparative method of fractionation, leading to the purification of four purified PIM6, corresponding to mono-, di-, tri-, and tetra-acyl forms. The structural analysis is further detailed below for the most complex entities, tri- and tetra-acyl forms. Glycosidic Analysis of Native Tetra-acyl Forms—The sequence of glycosyl residues in PIM6 was established using a range of high resolution NMR techniques applied to the tetra-acylated molecules. The 1H NMR spectrum of the native molecules in CDCl3/CD3OD/D2O, 60:35:8 (v/v/v), at 500 MHz showed a complex anomeric proton region (between 4.4 and 5.1 ppm) (Fig. 3). Anomeric signals were investigated thanks to the 1H-13C HMQC spectrum (Fig. 3). Indeed, from the protons resonating between 4.4 and 5.1 ppm, two of them correlated with carbons out of the anomeric zone: proton at δ 5.02 correlated with a carbon at 70.6 ppm and proton at δ 4.53 correlated with a carbon at 71.7 ppm. From previous studies (1Gilleron M. Ronet C. Mempel M. Monsarrat B. Gachelin G. Puzo G. J. Biol. Chem. 2001; 276: 34896-34904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 2Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (66) Google Scholar), they were respectively assigned to H2 of Gro and H3 of myo-Ins. These protons are deshielded due to the presence of gem acyl group. Moreover, the coupling constants measured on the proton at δ 4.53 (J 2,3 2.4 Hz and J 3,4 10.6 Hz) confirmed its attribution to myo-Ins H3. The other proton resonances correlating with carbons resonating around 100 ppm accounted for the six mannose units labeled from I to VI in decreasing order of their chemical shifts. Indeed, integration of the anomeric signals (H1) proved that the signal resonating at 4.88 ppm corresponded to two protons (II1 and III1). The α-anomeric configuration of the mannoses was deduced from the values of the one bound coupling constant (1 J C1,H1) above 170 Hz, measured on non-decoupled 1H-13C HMQC spectrum (not shown). The H2 protons of each α-Manp unit was determined using the COSY spectrum (Fig. 4A) although H2 of system I was partly hidden by the H3/H3′ of glycerol (Gro). The entire spin system of Gro can be analyzed from the COSY spectrum (Fig. 4A) and those of the myo-Ins from the HOHAHA spectrum (Fig. 4B). The six mannose spin systems could be completely assigned (Table I) using 1H-1H HOHAHA (Fig. 4B) and 1H-13C HMQC (Fig. 3). The deshielded values of the chemical shifts of the C2 of units I (δ 78.5) and IV (δ 79.1) and of C6 of units II (δ 65.8) and VI (δ 66.3) revealed the sites of glycosylation of the concerned units. The absence of deshielding concerning the carbons of systems III and V indicated that these units III and V were terminal. The chemical shift of the C6 of unit III (δ 63.9) (Table I) was intermediate between those of the C6 of units I, IV, and V (around 61.5 ppm) that were not glycosylated in C6 and those of the C6 of units II and VI (around 66.0) that were glycosylated in C6. Moreover, H6/H6′ protons of unit III were deshielded (4.02:4.15) (Table I). Thus, these data demonstrated that this unit is acylated in position 6.Table I1H and 13C NMR chemical shifts of the tetra-acylated forms measured at 308 K in CDCl3/CD3OD/D2O, 60:35:8, v/v/v123456ConclusionI5.023.833.643.393.483.47/3.642-O-Linked101.078.570.467.373.461.5II4.883.923.603.493.653.48/3.706-O-Linked101.870.170.967.070.565.8III4.883.843.583.473.784.02/4.15Terminal, 6-O-Acylated101.870.170.867.170.763.9IV4.863.703.683.423.423.51/3.602-O-Linked98.379.170.567.373.061.4V4.783.783.533.373.503.47/3.65Terminal102.370.471.067.471.061.5VI4.633.723.593.423.553.54/3.656-O-Linked99.770.370.867.370.966.3myo-Ins3.974.064.533.553.173.603-O-Acylated76.876.571.770.973.178.7Gro4.19/3.955.023.79/3.761,2-Di-O-Acylated62.970.663.6 Open table in a new tab The glycosidic sequence was next deduced from the inter-residual nuclear Overhauser effect contacts observed in the 1H-1H ROESY spectrum (Fig. 4C). This sequence was used here to observe short through space connectivities between the anomeric proton of each mannose and the proton of the adjacent glycosidically linked residue. The anomeric proton o" @default.
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• W2067539471 title "Acylation State of the Phosphatidylinositol Hexamannosides from Mycobacterium bovis Bacillus Calmette Guérin and Mycobacterium tuberculosis H37Rv and Its Implication in Toll-like Receptor Response" @default.
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