Tsukamurella paurometabola Lipoglycan, a New Lipoarabinomannan Variant with Pro-inflammatory Activity
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m310906200
ISSN1083-351X
AutoresKevin J. Gibson, Martine Gilleron, Patricia Constant, Thérèse Brando, Germain Puzo, Gurdyal S. Besra, Jérôme Nigou,
Tópico(s)Tuberculosis Research and Epidemiology
ResumoThe genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (α1→6)-Manp mannan domain, which is further substituted by an (α1→5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(α1→2)-Manp-(α1→ dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-α when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-α production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-α-inducing activity. This conclusively demonstrates that a linear (α1→6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (α1→6)-Manp mannan domain, which is further substituted by an (α1→5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(α1→2)-Manp-(α1→ dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-α when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-α production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-α-inducing activity. This conclusively demonstrates that a linear (α1→6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. There is a bewildering range of aerobic actinomycetes, found in almost any environment imaginable, with some pathogenic to humans and others that are not (1McNeil M.M. Brown J.M. Clin. Microbiol. Rev. 1994; 7: 357-417Crossref PubMed Scopus (568) Google Scholar). The aerobic actinomycetes may be further subdivided into the “nocardioform actinomycetes” (2Prauser H. Publ. Fac. Univ. Purkyne, Brno. K40. 1967; 196: 196-199Google Scholar). This informal terminology is now widely used to describe a number of organisms with similar characteristics, with key members including mycobacteria, nocardia, rhodococcus, and corynebacteria (3Lechevalier H.A. Williams S.T. Bergey's Manual of Systematic Bacteriology. 4. Williams & Wilkins, Baltimore, MD1986: 2348-2402Google Scholar). Unlike the previously mentioned members the genus Tsukamurella is in its infancy, whereas the type strain, Tsukamurella paurometabola, has had a long taxonomical history, with names including Corynebacterium paurometabolum (4Steinhaus E.A. J. Bacteriol. 1941; 42: 747-790Crossref Google Scholar), Gordona aurantiaca (5Tsukamura M. J. Gen. Microbiol. 1971; 68: 15-26Crossref PubMed Scopus (152) Google Scholar), and Rhodococcus aurantiacus (6Tsukamura M. Kawakami K. J. Clin. Microbiol. 1982; 16: 604-607Crossref PubMed Google Scholar). This taxonomical puzzle was finally resolved, when in 1988 Collins et al. (7Collins M.D. Smida J. Dorsch M. Stackenbrandt E. Int. J. Syst. Bacteriol. 1988; 38: 385-391Crossref Scopus (100) Google Scholar) showed that the 16 S RNAs of R. aurantiacus and C. paurometabolum were 99% homologous. And so they proposed reclassifying and renaming this organism T. paurometabola, after the microbiologist M. Tsukamura, who first isolated the species (7Collins M.D. Smida J. Dorsch M. Stackenbrandt E. Int. J. Syst. Bacteriol. 1988; 38: 385-391Crossref Scopus (100) Google Scholar). Cases of human infection with T. paurometabola are infrequent, nevertheless diagnosis rates are increasing, typically in patients with underlying predisposing factors, including immunosuppression (8Prinz G. Ban E. Fekete S. Szabo Z. J. Clin. Microbiol. 1985; 22: 472-474Crossref PubMed Google Scholar, 9Rey D. De Briel D. Heller R. Fraisse P. Partisani M. Leiva-Mena M. Lang J.M. AIDS. 1995; 9: 1379Crossref PubMed Scopus (7) Google Scholar), chronic pathology (6Tsukamura M. Kawakami K. J. Clin. Microbiol. 1982; 16: 604-607Crossref PubMed Google Scholar), and indwelling foreign bodies (10Shapiro C.L. Haft R.F. Gantz N.M. Doern G.V. Christenson J.C. O'Brien R. Overall J.C. Brown B.A. Wallace Jr., R.J. Clin. Infect. Dis. 1992; 14: 200-203Crossref PubMed Scopus (39) Google Scholar). However, there are a number of reported cases in which infected patients had no underlying factors, with Granel et al. (11Granel F. Lozniewski A. Barbaud A. Lion C. Dailloux M. Weber M. Schmutz J.L. Clin. Infect. Dis. 1996; 23: 839-840Crossref PubMed Scopus (25) Google Scholar) describing an inflammatory cutaneous infection in an otherwise healthy individual. All members of the nocardioform actinomycetes possess a similar whole cell carbohydrate composition, whereas the majority also contain long-chain branched fatty acids, termed mycolic acids (12Goodfellow M. Lechevalier M.P. Holt J.G. Bergey's Manual of Systematic Bacteriology. 4. Williams & Wilkins, Baltimore, MD1989: 2350-2361Google Scholar). The majority of our current knowledge about actinomycetes cell wall architecture comes from pioneering studies on mycobacterial strains (13Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar). Such work led to the identification, within the cell envelope, of a biosynthetically related family of glycolipids, phosphatidyl-myo-inositol mannosides (PIMs), 1The abbreviations used are: PIM, phosphatidyl-myo-inositol mannoside; Araf, arabinofuranose; APTS, 1-aminopyrene-3,6,8-trisulfonate; AsuLAM, amycolatopsis sulphurea lipoarabinomannan; αTpaLAM, α-mannosidase-treated TpaLAM; CE-LIF, capillary electrophoresis-laser-induced fluorescence; CE/ESI, capillary electrophoresis/electrospray ionization; dTpaLAM, deacylated TpaLAM; GC, gas chromatography; HMBC, heteronuclear multiple bound correlation spectroscopy; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; IL, interleukin; LAM, lipoarabinomannan; LM, lipomannan; mahTpaLAM, mild acidic-hydrolyzed TpaLAM; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; Manp, mannopyranose; ManLAM, LAM with mannosyl caps; MPI, mannosylphosphatidyl-myo-inositol; MS, mass spectrometry; PILAM, LAM with phosphoinositide caps; ReqLAM, rhodococcus equi lipoarabinomannan; t, terminal; TLR, toll-like receptor; TNF-α, tumor necrosis factor α; TpaLAM, Tsukamurella paurometabola lipoarabinomannan. and lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (13Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar). Mycobacterial LAM is a large heterogeneous macroamphiphile that possesses three distinct domains, a mannosylphosphatidyl-myo-inositol (MPI) anchor, a carbohydrate backbone, and various capping motifs (14Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (299) Google Scholar, 15Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (219) Google Scholar). The carbohydrate backbone is composed of two homopolysaccharides, D-mannan and D-arabinan. In all species described to date the D-mannan domain exists as a linear α(1→6)-Manp backbone substituted according to the species at the O-2 or O-3 positions by single Manp residues. The D-arabinan domain consists of a linear α(1→5)-Araf backbone punctuated by branching fashioned from 3,5-O-linked α-d-Araf residues (14Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (299) Google Scholar, 15Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (219) Google Scholar). In slow growing mycobacteria, such as Mycobacterium tuberculosis, the capping motifs consist of Manp residues (16Chatterjee D. Lowell K. Rivoire B. McNeil M.R. Brennan P.J. J. Biol. Chem. 1992; 267: 6234-6239Abstract Full Text PDF PubMed Google Scholar, 17Venisse A. Berjeaud J.M. Chaurand P. Gilleron M. Puzo G. J. Biol. Chem. 1993; 268: 12401-12411Abstract Full Text PDF PubMed Google Scholar); whereas fast growing mycobacteria, such as Mycobacterium smegmatis, possess phosphoinositol residues (18Gilleron 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 (91) Google Scholar, 19Khoo 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 (160) Google Scholar), resulting in LAM being termed either ManLAM or PILAM, respectively. In addition, a new class of LAMs has been described that lacks any capping motifs (20Guerardel 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 (108) Google Scholar), termed AraLAM. These subtle differences in the capping motifs are thought to explain the different immunomodulatory functions of ManLAM and PILAM. Indeed, a paradigm is emerging whereby ManLAMs possess the ability to inhibit the production of pro-inflammatory cytokines, such as interleukin-12 and TNF-α (21Knutson K.L. Hmama Z. Herrera-Velit P. Rochford R. Reiner N.E. J. Biol. Chem. 1998; 273: 645-652Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar); conversely, PILAM stimulates the production of such cytokines (18Gilleron 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 (91) Google Scholar, 23Adams L.B. Fukutomi Y. Krahenbuhl J.L. Infect. Immun. 1993; 61: 4173-4181Crossref PubMed Google Scholar). Lipoglycans from other nocardioform actinomycetes have been identified and have been shown to have related, but distinct structures to that of mycobacterial LAM. In particular, they are smaller in size and do not necessarily possess distinct mannan and arabinan domains (24Sutcliffe I.C. Arch. Oral Biol. 1995; 40: 1119-1124Crossref PubMed Scopus (27) Google Scholar, 25Garton N.J. Gilleron M. Brando T. Dan H.H. Giguere S. Puzo G. Prescott J.F. Sutcliffe I.C. J. Biol. 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Few of these studies examined whether these lipoglycans possessed any in vitro biological activity; nevertheless, in a recent publication we have demonstrated that a LAM-like molecule from Amycolatopsis sulphurea, designated AsuLAM, which possessed mannose capping motifs, failed to induce a pro-inflammatory cytokine pattern (27Gibson K.J.C. Gilleron M. Constant P. Puzo G. Nigou J. Besra G.S. Biochem. J. 2003; 372: 821-829Crossref PubMed Scopus (24) Google Scholar), in agreement with the findings that ManLAMs can modulate the immune response by inhibiting the induction of pro-inflammatory cytokines. We report here the isolation and structural characterization of a lipoglycan originating from T. paurometabola. Furthermore, we provide evidence for the molecular motifs underlying bacterial lipoglycan mediated pro-inflammatory cytokine responses. Bacteria and Growth Conditions—T. paurometabola, type strain DSM 20162, was purchased from Deutsche Sammlung van Mikroorganismen and Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures), Germany. It was routinely grown at 30 °C in GYM streptomyces medium, which contained 4 g of glucose, 4 g of yeast, and 10 g of maltose extract per liter of deionized water supplemented with 0.05% (w/v) Tween 80. Cells were grown to late log phase, harvested by centrifugation, washed, and lyophilized. Purification of TpaLAM—Purification procedures were adapted from protocols established for the extraction and purification of mycobacterial lipoglycans (31Nigou 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 (114) Google Scholar, 32Ludwiczak P. Gilleron M. Bordat Y. Martin C. Gicquel B. Puzo G. Microbiology. 2002; 148: 3029-3037Crossref PubMed Scopus (61) Google Scholar). Briefly, the cells were delipidated at 60 °C by mixing in CHCl3/CH3OH (1:1, v/v) overnight. The organic extract was removed by filtration, and the delipidated biomass was resuspended in deionized water and disrupted by sonication (MSE Soniprep, 12 micro amplitude, 60 s on, 90 s off for 10 cycles, on ice). The cellular glycans and lipoglycans were further extracted by refluxing the broken cells in 50% ethanol at 65 °C overnight. Contaminating proteins and glucans were removed by enzymatic degradation using protease and α-amylase treatments followed by dialysis. The resulting extract was resuspended in buffer A, 15% propan-1-ol in 50 mm ammonium acetate, and loaded onto an octyl-Sepharose CL-4B column (50 × 2.5 cm) and eluted with 400 ml of buffer A at 5 ml/h, enabling the removal of non-lipidic moieties (22Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar). The retained lipoglycans were eluted with 400 ml of buffer B, 50% propan-1-ol in 50 mm ammonium acetate. The resulting lipoglycans were resuspended in buffer C, 0.2 m NaCl, 0.25% sodium deoxycholate (w/v), 1 mm EDTA, and 10 mm Tris, pH 8, to a final concentration of 200 mg/ml and loaded onto a Sephacryl S-200 HR column (50 × 2.5 cm) and eluted with buffer C at a flow rate of 5 ml/h. Fractions (1.25 ml) were collected and analyzed by SDS-PAGE followed by periodic acid-silver nitrate staining. The resulting lipoglycan fractions were pooled, dialyzed extensively against water, lyophilized, and stored at –20 °C. Preparation of Chemically or Enzymatically Modified TpaLAM— Deacylated TpaLAM (dTpaLAM) was obtained by incubating 100 μg of TpaLAM with 200 μl of 0.1 n NaOH for 2 h at 37 °C. The reaction was stopped by extensive dialysis against water. TpaLAM lipomannan core (i.e. mild acid hydrolyzed TpaLAM, mahTpaLAM) was prepared by mild acid hydrolysis (0.1 m HCl at 110 °C for 25 min) of TpaLAM. TpaLAM lipomannan core was recovered after dialysis against water and analyzed for carbohydrate content via capillary electrophoresis coupled to laser-induced fluorescence (CE-LIF) as described below. α-Exomannosidase-treated TpaLAM (αTpaLAM) was prepared by incubating TpaLAM (100 μg) for 6 h at 37 °C in 30 μl of a jack bean α-mannosidase (Sigma) solution (2 mg/ml, 0.1 m sodium acetate buffer, pH 4.5, 1 mm ZnSO4). After a second addition of 50 μl of enzyme solution, the reaction was continued overnight. The reaction products were then dialyzed against 50 mm NH4CO3, pH 7.6. Elimination of α-mannosidase was achieved by denaturation (2 min at 110 °C) followed by overnight tryptic digestion (37 °C, 3.2 μg of trypsin). αTpaLAM was recovered after dialysis against water and analyzed for cap contents by CE-LIF (22Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (353) Google Scholar). MALDI-TOF/MS—The matrix used was 2,5-dihydroxybenzoic acid at a concentration of 10 μg/μl in a mixture of water/ethanol (1:1, v/v). 0.5 μl of TpaLAM, at a concentration of 10 μg/μl, was mixed with 0.5 μl of the matrix solution. Analyses were performed on a Voyager DE-STR MALDI-TOF instrument (PerSeptive Biosystems, Framingham, MA) using linear mode detection. Mass spectra were recorded in the negative mode using a 300-ns time delay with a grid voltage of 94% of full accelerating voltage (20 kV) and a guide wire voltage of 0.1%. The mass spectra were mass assigned using external calibration. Fatty Acid Analysis—200 μg of TpaLAM was deacylated using strong alkaline hydrolysis (200 μlof1 m NaOH at 110 °C for 2 h). The reaction mixture was neutralized with HCl, and the liberated fatty acids were extracted three times with chloroform and, after drying under nitrogen, were methylated using three drops of 10% (w/w) BF3 in methanol (Fluka) at 60 °C for 5 min. The reaction was stopped by the addition of water and the fatty acid methyl esters were extracted three times with chloroform. After drying, the fatty acid methyl esters were solubilized in 10 μl of pyridine and trimethylsilylated using 10 μl of hexamethyldisilazane and 5 μl of trimethylchlorosilane at room temperature for 15 min. After drying under a stream of nitrogen, the fatty acid derivatives were solubilized in cyclohexane before analysis by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Glycosidic Linkage Analysis—Glycosyl linkage composition was performed according to the modified procedure of Ciucanu and Kerek (33Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3206) Google Scholar). The per-O-methylated TpaLAM was hydrolyzed using 500 μl of 2 m trifluoroacetic acid at 110 °C for 2 h, reduced using 350 μl of a 10 mg/ml solution of NaBD4 (1 m NH4OH/C2H5OH, 1:1, v/v) and per-O-acetylated using 300 μl of acetic anhydride for 1 h at 110 °C. The resulting alditol acetates were solubilized in cyclohexane before analysis by GC and GC/MS. APTS Derivatization—1–5 μg of lipoglycans, in the presence of mannoheptose as an internal standard, were hydrolyzed using either strong acid hydrolysis (30 μl of 2 m trifluoroacetic acid at 110 °C for 2 h) (total carbohydrate analysis) or mild acid hydrolysis (30 μl of 0.1 m HCl at 110 °C for 20 or 30 min) (caps analysis). The samples were dried and mixed with 0.3 μl of 0.2 m 1-aminopyrene-3,6,8-trisulfonate (APTS) (Interchim, Montluçon, France) in 15% acetic acid and 0.3 μl of a 1 m sodium cyanoborohydride solution dissolved in tetrahydrofuran. The reaction mixture was heated at 55 °C for 90 min and subsequently quenched by the addition of 20 μl of water. A 2-μl solution of the APTS derivatized solution was diluted 10-fold before being subjected to capillary electrophoresis. CE-LIF—Analyses were performed on a P/ACE capillary electrophoresis system (Beckman Instruments, Inc.) with the cathode on the injection side and the anode on the detection side (reverse polarity). The electropherograms were acquired and stored on a Dell XPS P60 computer using the System Gold software package (Beckman Instruments, Inc.). APTS derivatives were loaded by applying 0.5 p.s.i. (3.45 kPa) vacuum for 5 s (6.5 nl injected). Separations were performed using an uncoated fused-silica capillary column (Sigma, Division Supelco, Saint-Quentin-Fallavier, France) of 50-μm internal diameter with 40 cm of effective length (47-cm total length). Analyses were carried out at a temperature of 25 °C with an applied voltage of 20 kV using acetic acid 1% (w/v), triethylamine 30 mm in water, pH 3.5, as running electrolyte. The detection system consisted of a Beckman laser-induced fluorescence (LIF) equipped with a 4-milliwatt argon-ion laser with the excitation wavelength of 488 nm and emission wavelength filter of 520 nm. CE-ESI/MS—Analyses were performed on a CE system P/ACE™ MDQ (Beckman Coulter, Inc) with a 75-μm × 80-cm fused-silica capillary. The outlet was integrated into the electrospray ionization (ESI) needle that was directly coupled to an ion trap MS system (LCQ™ DUO, ThermoFinnigan, Inc.). Separations were carried out with an electrolyte composed of acid acetic (1%, v/v), triethylamine (30 mm), pH 3.5, and an applied voltage of –20 kV. Migration was monitored by the total ion current. During analysis, temperature was constantly maintained (25 °C) along the capillary, and the outlet end of the capillary was at a spray voltage of 4 kV. The sheath liquid, consisting of water: isopropanol (20:80), was infused to the ESI needle through a syringe pump at a flow rate of 3 μl min–1 using nitrogen as a nebulizing gas. For measurements, negative mode was used and all data were collected and analyzed on Xcalibur software. NMR Spectroscopy—NMR spectra were recorded on a Bruker DMX-500 spectrometer equipped with a double resonance (1H/X)-BBi z-gradient probe head. TpaLAM (10 mg) was exchanged in D2O (D, 99.97% from Euriso-top, Saint-Aubin, France), with intermediate lyophilization, then re-dissolved in 0.4 ml of Me2SO-d6 (D, 99.8% from Euriso-top, Saint-Aubin, France) and analyzed in 200 × 5 mm 535-PP NMR tubes at 343 K. Data were processed on a Bruker-X32 workstation using the xwinnmr program. Proton and carbon chemical shifts are expressed in ppm and referenced relative to internal Me2SO signals at δH 2.52 and δC 40.98. The one dimensional (1D) proton (1H) spectrum was recorded using a 90° tipping angle for the pulse and 1 s as recycle delay between each of the 128 acquisitions of 1.64 s. The spectral width of 2948 Hz was collected in 16,000 complex points that were multiplied by a Gaussian function (LB = –1, GB = 0.2) prior to processing to 32,000 real points in the frequency domain. After Fourier transformation, the spectra were baseline-corrected with a fourth order polynomial function. Two-dimensional (2D) spectra were recorded without sample spinning, and data were acquired in the phase-sensitive mode using the time-proportional phase increment (TPPI) method. The 2D 1H-13C heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) were recorded in the proton-detected mode with a Bruker 5-mm 1H broad band tunable probe with reverse geometry. The Globally optimized Alternating-phase Rectangular Pulses sequence (34Shaka A.J. Barker P.B. Freeman R. J. Magn. Reson. 1985; 64: 547-552Google Scholar) at the carbon frequency was used as a composite pulse decoupling during acquisition. The 1H-13C HMQC spectrum was obtained according to Bax and Subramanian pulse sequence (35Bax A. Subramanian S. J. Magn. Reson. 1986; 67: 565-569Google Scholar). Spectral widths of 25,154 Hz in 13C and 2,948 Hz in 1H dimensions were used to collect a 2,048 × 413 (TPPI) point data matrix with 80 scans/t1 value expanded to 4,096 × 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by π /2 was applied in both dimensions. The 1H-13C HMBC spectrum was obtained using the Bax and Summers pulse sequence (36Bax A. Summers M.F. J. Am. Chem. Soc. 1986; 108: 2093-2094Crossref Scopus (3272) Google Scholar). Spectral widths of 25,154 Hz in 13C and 2,948 Hz in 1H dimensions were used to collect a 2,048 × 512 (TPPI) point data matrix with 96 scans/t1 value expanded to 4,096 × 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by π /2 was applied in both dimensions. The 2D 1H-1H HOHAHA spectrum was recorded using a MLEV-17 mixing sequence of 112 ms (37Bax A. Davies D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar). The spectral width was 2,948 Hz in both F2 and F1 dimensions. 512 spectra of 4,096 data points with 24 scans/t1 increment were recorded. TNF-α Production by Macrophages—THP-1 and J774 monocyte/macrophage human and murine cell lines, respectively, were maintained in continuous culture with RPMI 1640 medium (Invitrogen), 10% fetal calf serum (Invitrogen) in an atmosphere of 5% CO2 at 37 °C, THP-1 as non-adherent and J774 as adherent cells. Purified native or modified TpaLAM were added in duplicate or triplicate to monocyte/macrophage cells (5 × 105 cells/well) in 24-well culture plates and then incubated for 20 h at 37 °C. Stimuli were previously incubated for 1 h at 37 °C in the presence or absence of 10 μg/ml polymyxin B (Sigma) known to inhibit the effect of (contaminating) LPS (23Adams L.B. Fukutomi Y. Krahenbuhl J.L. Infect. Immun. 1993; 61: 4173-4181Crossref PubMed Google Scholar). To investigate the TLR dependence of TNF-α-inducing TpaLAM activity, monoclonal anti-TLR-2 (clone TL2.1, eBioscience) or anti-TLR-4 (clone HTA125, Serotec) antibodies or an IgG2a isotype control (clone eBM2a, eBioscience) at a concentration of 10 μg/ml were added together with TpaLAM to THP-1 cells. Supernatants from THP-1 cells were assayed for TNF-α by sandwich enzyme-linked immunosorbent assay using commercially available kits and according to manufacturer's instructions (R&D Systems). Supernatants from J774 cells were assayed for TNF-α using the previously described cytotoxic assay against WEHI164 clone 13 cells (38Espevik T. Nissen-Meyer J. J. Immunol. Methods. 1986; 95: 99-105Crossref PubMed Scopus (1280) Google Scholar). Basically, 50 μl of supernatant was added to 50 μlof WEHI cells (5 × 105 cells/ml) in flat-bottom 96-well plates and incubated for 20 h at 37 °C, then 50 μl of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 1 mg/ml in phosphate-buffered saline) were added to each well and incubated for 4 h. Formazan crystals were solubilized with 100 μl of lysis buffer (N,N-dimethyl formamide, 30% SDS (1:2) adjusted to pH 4.7 with acetic acid), and the optical density was read at 570 nm with an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments). The TNF-α content of supernatants was determined by comparing to a reference curve obtained using serial dilutions of human recombinant TNF-α (Invitrogen). LPS was from Escherichia coli 055:B5 (Sigma) and Man-LAM from Mycobacterium bovis BCG. A lipoglycan with a SDS-PAGE migration similar to that of M. tuberculosis LAM was purified from T. paurometabola (Fig. 1A). The negative MALDI mass spectrum of the lipoglycan exhibited a broad unresolved peak centered at m/z 12500, indicating a molecular mass around 12.5 kDa for the major molecular species of this lipoglycan (Fig. 1B). CE-LIF analysis of the total acid hydrolyzed lipoglycan showed that it contained Ara and Man, in a ratio of 1:1.7. In addition, myo-inositol, glycerol, and fatty acids were also detected by GC analysis. The predominant fatty acids identified were palmitic (C16:0, 50%) and octadecenoic (C18:1, 20%), with smaller amounts of stearic (C18:0, 15%) and tuberculostearic (methyl-10-methyloctadecanoic, C19:0, 15%) acids. Altogether, the lipoglycan exhibited the basic components of a structure related to mycobacterial LAM and was subsequently termed TpaLAM. NMR Signal Assignment—Per-O-methylation analysis of TpaLAM indicated the presence of 5-Araf, t-Manp, 2,5-Araf, and 2-Manp residues in similar ratios, with slightly less 6-Manp detectable. Accordingly, the 1H-NMR anomeric region of TpaLAM exhibited five anomeric signals at δ 5.07 (I1), δ 5.02 (II1), δ 4.94 (III1), δ 4.85 (IV1), and δ 4.71 (V1), in a ratio 1.4/1.5/1.8/1.5/1 (Fig. 2A). As revealed by the 1H-13C HMQC spectrum, their corresponding anomeric carbons resonate at δ 99.5 (I1), δ 107.1 (II1), δ 103.1 (III1), δ 109.3 (IV1), and δ 100.7 (V1), respectively (Fig. 2D). Proton and carbon resonances of the different spin systems were assigned from 1H-13C HMQC and 1H-1H HOHAHA experiments (partially shown in Figs. 2C, 2D, and Fig. 3, respectively) and were based on our previous studies with mycobacterial LAMs (17Venisse A. Berjeaud J.M. Chaurand P. Gilleron M. Puzo G. J. Biol. Chem. 1993; 268: 12401-12411Abstract Full Text PDF PubMed Google Scholar, 39Gilleron M. Bala L. Br
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