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A Lipomannan Variant with Strong TLR-2-dependent Pro-inflammatory Activity in Saccharothrix aerocolonigenes

2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês

10.1074/jbc.m505498200

ISSN

1083-351X

Autores

Kevin J. Gibson, Martine Gilleron, Patricia Constant, Bénédicte Sichi, Germain Puzo, Gurdyal S. Besra, Jérôme Nigou,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

Lipomannans (LMs) are powerful pro-inflammatory lipoglycans found in mycobacteria and related genera, however the molecular bases of their activity are not fully understood. We report here the isolation and the structural and functional characterization of a new lipomannan variant present in the Pseudonocardineae, Saccharothrix aerocolonigenes, designated SaeLM. Using a range of chemical degradations, NMR experiments, and mass spectrometry analyses, SaeLM revealed a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by an original carbohydrate structure whereby an (α1→6)-Manp backbone is substituted at >80% of the O-2 position by side chains composed of Manp-(α1→2)-Manp-(α1→. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicated a distribution of SaeLM glyco-forms ranging from 19 to 61 Manp units, which centered on species containing 37 or 40 Manp units. SaeLM induced a Toll-like receptor 2 (TLR-2)-dependent production of tumor necrosis factor-α (TNF-α) by human THP-1 monocyte/macrophage cell lines and interestingly was found to be the strongest inducer of this pro-inflammatory cytokine when compared with other LAM/LM-like molecules. We previously established that a linear (α1→6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. We demonstrate here that by adding side chains and increasing their size, one may potentiate this activity. These findings should enable a better understanding of the structure/function relationships of TLR-2-dependent lipoglycan signaling. Lipomannans (LMs) are powerful pro-inflammatory lipoglycans found in mycobacteria and related genera, however the molecular bases of their activity are not fully understood. We report here the isolation and the structural and functional characterization of a new lipomannan variant present in the Pseudonocardineae, Saccharothrix aerocolonigenes, designated SaeLM. Using a range of chemical degradations, NMR experiments, and mass spectrometry analyses, SaeLM revealed a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by an original carbohydrate structure whereby an (α1→6)-Manp backbone is substituted at >80% of the O-2 position by side chains composed of Manp-(α1→2)-Manp-(α1→. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicated a distribution of SaeLM glyco-forms ranging from 19 to 61 Manp units, which centered on species containing 37 or 40 Manp units. SaeLM induced a Toll-like receptor 2 (TLR-2)-dependent production of tumor necrosis factor-α (TNF-α) by human THP-1 monocyte/macrophage cell lines and interestingly was found to be the strongest inducer of this pro-inflammatory cytokine when compared with other LAM/LM-like molecules. We previously established that a linear (α1→6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. We demonstrate here that by adding side chains and increasing their size, one may potentiate this activity. These findings should enable a better understanding of the structure/function relationships of TLR-2-dependent lipoglycan signaling. Lipomannan (LM) 1The abbreviations used are: LM, lipomannan; Araf, arabinofuranose; AraLAM, non-capped lipoarabinomannan; dSaeLM, deacylated S. aerocolonigenes lipomannan; GC, gas chromatography; HMBC, heteronuclear multiple bound correlation spectroscopy; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; IL, interleukin; LAM, lipoarabinomannan; 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; PILAM, LAM with phosphoinositide caps; PIM, phosphatidyl-myo-inositol mannoside; ROESY, rotating Overhauser and exchange spectroscopy; SaeLM, S. aerocolonigenes lipomannan; t, terminal; TLR, Toll-like receptor; TNF-α, tumor necrosis factor α; TpaLAM, T. paurometabola lipoarabinomannan; BCG, bacillus Calmette-Guerin; nOe, nuclear Overhauser effect. and lipoarabinomannan (LAM) are related powerful immunomodulatory lipoglycans found in mycobacterial cell walls (1.Chatterjee D. Khoo K.H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (301) Google Scholar, 2.Gilleron M. Rivière M. Puzo G. Aubery M. Glycans in Cell Interaction and Recognition: therapeutic Aspects. Harwood Academic Press, Amsterdam2001: 113-140Google Scholar, 3.Nigou J. Gilleron M. Rojas M. Garcia L.F. Thurnher M. Puzo G. Microbes Infect. 2002; 4: 945-953Crossref PubMed Scopus (130) Google Scholar, 4.Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (229) Google Scholar, 5.Briken V. Porcelli S.A. Besra G.S. Kremer L. Mol. Microbiol. 2004; 53: 391-403Crossref PubMed Scopus (361) Google Scholar). Their structures originate from a phosphatidyl-myo-inositol (MPI) anchor, which is mannosylated to generate LM (4.Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (229) Google Scholar, 6.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 (147) Google Scholar) and further arabinosylated to give LAM. The non-reducing termini of the arabinosyl side chains can be substituted by capping motifs, yielding to the classification of LAM into three families. LAM from slow growing mycobacteria bearing mannose caps, i.e. mono- or (α1→2)-di- or tri-mannoside units, are designated as ManLAM. In contrast, LAM from fast growing mycobacteria capped by phospho-myo-inositol units or not capped at all are termed PILAM and AraLAM, respectively (4.Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (229) Google Scholar). LAM and LM exhibit a broad spectrum of immunomodulatory activities, including the ability to modulate the production of macrophage-derived Th1 pro-inflammatory cytokines, most commonly TNF-α and IL-12. For example, ManLAM are able to inhibit the LPS-induced production of IL-12 and TNF-α (7.Knutson 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 (137) Google Scholar, 8.Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (358) Google Scholar). So, ManLAM contributes, via an immunosuppressive effect, to the persistence of slow-growing mycobacteria in the human reservoir. ManLAM anti-inflammatory activity has also been shown to require the interaction of ManLAM with the mannose receptor and/or dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin via the mannose capping motifs (8.Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. J. Immunol. 2001; 166: 7477-7485Crossref PubMed Scopus (358) Google Scholar, 9.Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (911) Google Scholar, 10.Tailleux L. Maeda N. Nigou J. Gicquel B. Neyrolles O. Trends Microbiol. 2003; 11: 259-263Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In contrast, PILAM are able to induce the release of a variety of pro-inflammatory cytokines through the activation of Toll-like receptors 2 (TLR-2) (11.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 (92) Google Scholar, 12.Adams L.B. Fukutomi Y. Krahenbuhl J.L. Infect. Immun. 1993; 61: 4173-4181Crossref PubMed Google Scholar, 13.Means T.K. Lien E. Yoshimura A. Wang S. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 6748-6755Crossref PubMed Google Scholar). This activity is likely to require PI caps, because AraLAM does not show any activity (14.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 (111) Google Scholar). Early studies demonstrated that LM from Mycobacterium sp. induce expression of pro-inflammatory cytokines (15.Barnes P.F. Chatterjee D. Abrams J.S. Lu S. Wang E. Yamamura M. Brennan P.J. Modlin R.L. J. Immunol. 1992; 149: 541-547Crossref PubMed Google Scholar). A set of recent reports have shown that LM from both pathogenic and non-pathogenic mycobacterial species, independent of their origin, were potent stimulators of TNF-α, IL-8, and IL-12 (16.Vignal C. Guerardel Y. Kremer L. Masson M. Legrand D. Mazurier J. Elass E. J. Immunol. 2003; 171: 2014-2023Crossref PubMed Scopus (119) Google Scholar, 17.Quesniaux V.J. Nicolle D.M. Torres D. Kremer L. Guerardel Y. Nigou J. Puzo G. Erard F. Ryffel B. J. Immunol. 2004; 172: 4425-4434Crossref PubMed Scopus (217) Google Scholar, 18.Dao D.N. Kremer L. Guerardel Y. Molano A. Jacobs Jr., W.R. Porcelli S.A. Briken V. Infect. Immun. 2004; 72: 2067-2074Crossref PubMed Scopus (138) Google Scholar). Furthermore, LM was shown to activate macrophages in a TLR-2-dependent, and TLR-4- and TLR-6-independent manner (16.Vignal C. Guerardel Y. Kremer L. Masson M. Legrand D. Mazurier J. Elass E. J. Immunol. 2003; 171: 2014-2023Crossref PubMed Scopus (119) Google Scholar, 17.Quesniaux V.J. Nicolle D.M. Torres D. Kremer L. Guerardel Y. Nigou J. Puzo G. Erard F. Ryffel B. J. Immunol. 2004; 172: 4425-4434Crossref PubMed Scopus (217) Google Scholar, 18.Dao D.N. Kremer L. Guerardel Y. Molano A. Jacobs Jr., W.R. Porcelli S.A. Briken V. Infect. Immun. 2004; 72: 2067-2074Crossref PubMed Scopus (138) Google Scholar). The ManLAM/LM balance might thus be a parameter influencing the net immune response against mycobacteria. Indeed, according to their activity, lipoglycans are therefore likely to favor either the persistence or the killing of the corresponding mycobacteria (3.Nigou J. Gilleron M. Rojas M. Garcia L.F. Thurnher M. Puzo G. Microbes Infect. 2002; 4: 945-953Crossref PubMed Scopus (130) Google Scholar). Induction of a protective pro-inflammatory response via TLR signaling should be to the benefit of the host (19.Heldwein K.A. Liang M.D. Andresen T.K. Thomas K.E. Marty A.M. Cuesta N. Vogel S.N. Fenton M.J. J. Leukoc. Biol. 2003; 74: 277-286Crossref PubMed Scopus (180) Google Scholar, 20.Heldwein K.A. Fenton M.J. Microbes Infect. 2002; 4: 937-944Crossref PubMed Scopus (136) Google Scholar, 21.Basu S. Fenton M.J. Am. J. Physiol. 2004; 286: L887-L892Crossref PubMed Scopus (170) Google Scholar), whereas stimulation of anti-inflammatory response via mannose receptor or dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin should be to the benefit of the pathogen (3.Nigou J. Gilleron M. Rojas M. Garcia L.F. Thurnher M. Puzo G. Microbes Infect. 2002; 4: 945-953Crossref PubMed Scopus (130) Google Scholar, 10.Tailleux L. Maeda N. Nigou J. Gicquel B. Neyrolles O. Trends Microbiol. 2003; 11: 259-263Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The molecular bases of LM/LAM pro-versus anti-inflammatory activities are not yet fully understood. Nevertheless, it seems clear that LM or the lipomannan moiety of LAM bear the intrinsic capacity to induce the production of TNF-α and IL-12 (16.Vignal C. Guerardel Y. Kremer L. Masson M. Legrand D. Mazurier J. Elass E. J. Immunol. 2003; 171: 2014-2023Crossref PubMed Scopus (119) Google Scholar, 22.Gibson K.J. Gilleron M. Constant P. Brando T. Puzo G. Besra G.S. Nigou J. J. Biol. Chem. 2004; 279: 22973-22982Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, the presence of the arabinan moiety on LAM inhibits the pro-inflammatory activity, presumably by masking the mannan core (23.Sidobre S. Nigou J. Puzo G. Riviere M. J. Biol. Chem. 2000; 275: 2415-2422Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 24.Sidobre S. Puzo G. Riviere M. Biochem. J. 2002; 365: 89-97Crossref PubMed Scopus (34) Google Scholar) and thus limiting its accessibility to the TLR (16.Vignal C. Guerardel Y. Kremer L. Masson M. Legrand D. Mazurier J. Elass E. J. Immunol. 2003; 171: 2014-2023Crossref PubMed Scopus (119) Google Scholar, 22.Gibson K.J. Gilleron M. Constant P. Brando T. Puzo G. Besra G.S. Nigou J. J. Biol. Chem. 2004; 279: 22973-22982Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Also, the type of capping motifs may then direct LAM activity toward a pro-(PILAM) or anti-(ManLAM) inflammatory activity (3.Nigou J. Gilleron M. Rojas M. Garcia L.F. Thurnher M. Puzo G. Microbes Infect. 2002; 4: 945-953Crossref PubMed Scopus (130) Google Scholar). Further insights into deciphering these complex molecular interactions could benefit from the structural and functional characterization of LAM variants. Indeed, lipoglycans are not restricted to members of the mycobacteria, and a number of non-mycobacterial lipoglycans have been isolated and characterized in several actinomycete genera, including Rhodococcus (25.Garton N.J. Gilleron M. Brando T. Dan H.H. Giguere S. Puzo G. Prescott J.F. Sutcliffe I.C. J. Biol. Chem. 2002; 277: 31722-31733Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 26.Gibson K.J. Gilleron M. Constant P. Puzo G. Nigou J. Besra G.S. Microbiology. 2003; 149: 1437-1445Crossref PubMed Scopus (28) Google Scholar), Gordonia (27.Flaherty C. Sutcliffe I.C. Syst. Appl. Microbiol. 1999; 22: 530-533Crossref PubMed Scopus (23) Google Scholar), Amycolatopsis (28.Gibson K.J. Gilleron M. Constant P. Puzo G. Nigou J. Besra G.S. Biochem. J. 2003; 372: 821-829Crossref PubMed Scopus (24) Google Scholar), Corynebacterium (29.Sutcliffe I.C. Arch. Oral. Biol. 1995; 40: 1119-1124Crossref PubMed Scopus (27) Google Scholar), and most recently Turicella (30.Gilleron M. Garton N.J. Nigou J. Brando T. Puzo G. Sutcliffe I.C. J. Bacteriol. 2005; 187: 854-861Crossref PubMed Scopus (30) Google Scholar) and Tsukamurella (22.Gibson K.J. Gilleron M. Constant P. Brando T. Puzo G. Besra G.S. Nigou J. J. Biol. Chem. 2004; 279: 22973-22982Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In the latter study, we demonstrated that LAM from Tsukamurella paurometabola (TpaLAM) possesses a similar structural prototype when compared with mycobacterial LAM, with distinct mannan and arabinan domains, yet had weak biological activity (22.Gibson K.J. Gilleron M. Constant P. Brando T. Puzo G. Besra G.S. Nigou J. J. Biol. Chem. 2004; 279: 22973-22982Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar); however, upon chemical degradation of the arabinan domain, the resulting lipomannan moiety elicited a powerful pro-inflammatory response as previously demonstrated with ManLAM (16.Vignal C. Guerardel Y. Kremer L. Masson M. Legrand D. Mazurier J. Elass E. J. Immunol. 2003; 171: 2014-2023Crossref PubMed Scopus (119) Google Scholar). Interestingly, the TpaLAM lipomannan moiety is composed of a linear (α1→6)-Manp chain linked to the MPI anchor demonstrating that this structure alone is sufficient in providing pro-inflammatory activity, and that, importantly, the branched t-Manp units are not necessarily required (22.Gibson K.J. Gilleron M. Constant P. Brando T. Puzo G. Besra G.S. Nigou J. J. Biol. Chem. 2004; 279: 22973-22982Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In the present study we report the isolation, structural, and functional characterization of a LM molecule from the Pseudonocardineae, Saccharothrix aerocolonigenes (31.Stackebrandt E. Rainey F.A. Ward-Rainey N.L. Int. J. Syst. Bacteriol. 1997; 47: 479-491Crossref Scopus (1345) Google Scholar). The investigation revealed an original structure, and, furthermore, we demonstrate that LM possessed potent pro-inflammatory activity. As such, the structure/function relationship of the lipomannan is discussed, enabling further insights into the molecular basis of lipoglycan-mediated inflammatory responses. Bacteria and Growth Conditions—S. aerocolonigenes, type strain DSM 40034 (S. aerocolonigenes subsp. aerocolonigenes, recently renamed as Lechevaliera aerocolonigenes (32.Labeda D.P. Hatano K. Kroppenstedt R.M. Tamura T. Int. J. Syst. Evol. Microbiol. 2001; 51: 1045-1050Crossref PubMed Scopus (62) Google Scholar)) was purchased from DSMZ, Germany. It was routinely grown at 30 °C in GYM streptomyces medium, which contained 4 g of glucose, 4 g of yeast extract, and 10 g of maltose per liter of deionized water supplemented with 0.05% (w/v) Tween 80. Cells were grown to late log phase and harvested by centrifugation, washed, and lyophilized. Purification of SaeLM—Purification procedures were adapted from protocols established for the extraction and purification of mycobacterial lipoglycans (33.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 (116) Google Scholar, 34.Ludwiczak 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 then 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. 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 Deacylated SaeLM—Deacylated SaeLM (dSaeLM) was obtained by incubating 100 μg of SaeLM with 200 μl of 0.1 n NaOH for 2 h at 37 °C. The reaction was stopped by extensive dialysis against water. MALDI/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 SaeLM, 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 95% of full accelerating voltage (24 kV) and a guide wire voltage of 0.05%. The mass spectra were mass assigned using external calibration. Fatty Acid Analysis—200 μg of SaeLM was deacylated using strong alkaline hydrolysis (200 μl of 1 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 trimethyl-silylated using 10 μl of hexamethyl-disilazane 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 (35.Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3281) Google Scholar). The per-O-methylated SaeLM 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 (NH4OH 1 m/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. NMR Spectroscopy—NMR spectra were recorded on a Bruker DMX-500 spectrometer equipped with a double resonance (1H/X)-BBi z-gradient probe head. SaeLM (20 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), 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) spectra were recorded using a 90° tipping angle for the pulse and 1 s as recycle delay between each of the 387 acquisitions of 1.64 s. The spectral width of 3,064 Hz was collected in 16,000 complex points that were multiplied by a Gaussian function (LB = -1, GB = 0.4) prior to processing to 32,000 real points in the frequency domain. After Fourier transformation, the spectra were base-line corrected with a fourth order polynomial function. The 1D 31P spectrum was measured at 202.46 MHz at 343 K and phosphoric acid (85%) was used as external reference (δP 0.0). The spectral width of 20 kHz was collected in 16,000 complex points that were multiplied by an exponential function (LB = 1 Hz) prior to processing to 32,000 real points in the frequency domain. The scan number was 256. Two-dimensional (2D) spectra were recorded without sample spinning, and data were acquired in the phase-sensitive mode using the time-proportional phase increment method. The 2D 1H-13C Heteronuclear Multiple Quantum Correlation (HMQC) and 1H-31P HMQC-HOHAHA 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 (GARP) sequence (36.Shaka A.J. Barker P.B. Freeman R. J. Magn. Reson. 1985; 64: 547-552Google Scholar) at the carbon or phosphorus frequency was used as a composite pulse decoupling during acquisition. The 1H-13C HMQC spectrum was obtained according to Bax and Subramanian pulse sequence (37.Bax A. Subramanian S. J. Magn. Reson. 1986; 67: 565-569Google Scholar). Spectral widths of 25,154 Hz in 13C and 2200 Hz in 1H dimensions were used to collect a 2,048 × 512 (time-proportional phase increment) point data matrix with 56 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. A 1H-13C HMBC spectrum was obtained using the Bax and Summers pulse sequence (38.Bax A. Summers M.F. J. Am. Chem. Soc. 1986; 108: 2093-2094Crossref Scopus (3287) Google Scholar). Spectral widths of 25,154 Hz in 13C and 3,064 Hz in 1H dimensions were used to collect a 2,048 × 480 (time-proportional phase increment) 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. A 1H-31P HMQC-HOHAHA spectrum was obtained using the Lerner and Bax pulse sequence (39.Lerner L. Bax A. J. Magn. Reson. 1986; 69: 375-380Google Scholar). Spectral widths of 1,620 Hz in 31P and 3,064 Hz in 1H dimensions were used to collect a 2,048 × 80 (time-proportional phase increment) point data matrix with 16 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 110 ms (40.Bax A. Davies D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar). The spectral width was 3,064 Hz in both F2 and F1 dimensions. 450 spectra of 4,096 data points with 24 scans/t1 increment were recorded. The 2D 1H-1H ROESY spectrum was acquired at a mixing time of 300 ms (41.Bax A. Davis D.G. J. Magn. Reson. 1985; 63: 207-213Google Scholar). The spectral width was 3,064 Hz in both dimensions. 512 spectra of 2,048 data points with 24 scans/t1 increment were recorded. TNF-α Production by Macrophages—A THP-1 monocyte/macrophage human cell line was maintained in continuous culture with RPMI 1640 medium (Invitrogen), 10% fetal calf serum (Invitrogen) in an atmosphere of 5% CO2 at 37 °C, as non-adherent cells. Purified native or modified SaeLM as well as the other stimuli 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 (12.Adams 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 SaeLM 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 concentrations of 10 and 20 μg/ml were added together with SaeLM 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 the manufacturer's instructions (R&D Systems). LPS was from Escherichia coli 055:B5 (Sigma), ManLAM and LM were from Mycobacterium bovis BCG, and mahTpaLAM was from T. paurometabola (22.Gibson K.J. Gilleron M. Constant P. Brando T. Puzo G. Besra G.S. Nigou J. J. Biol. Chem. 2004; 279: 22973-22982Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). A lipoglycan that migrated in SDS-PAGE between Mycobacterium tuberculosis ManLAM and LM was purified from S. aerocolonigenes (Fig. 1). The lipoglycan contained mannose as the sole carbohydrate, myo-inositol, glycerol, and fatty acids. The predominant fatty acids identified by GC were 14-methylpentadecanoic (iC16:0) (33%), palmitic (C16:0) (20%), and octadecenoic (C18:1) (33%), with smaller amounts of stearic (C18:0) (6%), and various isomers of heptadecanoic (C17) (altogether 8%) acids. The lipoglycan exhibited the same basic components of a structure related to mycobacterial LM and was subsequently termed SaeLM. Mannan Backbone—The 1H NMR anomeric region of SaeLM was dominated by three signals at δ 5.03 (Ia1), δ 4.98 (IIa1), and δ 4.84 (IIIa1), with nearly identical intensities (Fig. 2A). As revealed by the 1H-13C HMQC spectrum, their corresponding anomeric carbons resonate at δ 101.4 (Ia1), δ 102.4 (IIa1), and δ 99.4 (IIIa1) (Fig. 2E). Anomeric proton and carbon resonances of the different spin systems were assigned from 2D 1H-13C HMQC (Fig. 2, D and E) and HMBC (Fig. 2C) and 1H-1H HOHAHA (Fig. 3C) and ROESY (Fig. 3D) experiments based on our previous studies with mycobacterial LAMs and LMs (42.Gilleron M. Bala L. Brando T. Vercellone A. Puzo G. J. Biol. Chem. 2000; 275: 677-684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 43.Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (67) Google Scholar). The assignments are summarized in Table I. Spin systems Ia, IIa, and IIIa were unambiguously assigned to 2-α-Manp, t-α-Manp, and 2,6-α-Manp, respectively, in agreement with per-O-methylation data. The α-anomeric configuration was demonstrated through the magnitude of their 1JH1,C1 coupling constants determined as 174, 171, and 169 Hz for spin systems Ia, IIa, and IIIa, respectively (α-O-Me-Manp: 1JH-1,C-1 170 Hz; β-O-Me-Manp: 1JH-1,C-1 161 Hz (44.Bock K. Pedersen C.J. J. Chem. Soc. Perkin Transac. 1974; 2: 293Crossref Google Scholar)). The pyranose ring was deduced from their δC-5 at 74.7, 73.1, and 74.2, respectively, and the presence on the 1H-13C HMBC spectrum (Fig. 2C) of cross-peaks between their H-1 and their respective C-5. 1H and 13C chemical shifts of spin system IIa (Table I) indicated an unsubstituted t-Manp unit (42.Gilleron M. Bala L. Brando T. Vercellone A. Puzo G. J. Biol. Chem. 2000; 275: 677-684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 43.Gilleron M. Nigou J. Cahuzac B. Puzo G. J. Mol. Biol. 1999; 285: 2147-2160Crossref PubMed Scopus (67) Google Scholar). Glycosylation at position 2 of 2-α-Manp (I) and 2,6-α-Manp (III) units was determined by the deshielding of their C-2 resonance at δ 77.0 and δ 77.8, respectively, as compared with the C-2 resonance of the unsubstituted t-α-Manp unit at δ 71.0 (Δδ 6.0 and 6.8 ppm, respectively) (Fig. 2D and Table I). Glycosylation at position 6 of 2,6-α-Manp (III) units was shown through the deshielding of the C-6 resonance at δ 66.0 as compared with the C-6 resonance of the unsubstituted t-α-Manp unit at δ 62.4 (Δδ 3.6 ppm) (Fig. 2D and Table I).Fig. 31D 1H (A and B) and 2D 1H-1H HOHAHA τm 110 ms (C) and ROESY τm 300 ms (D) spectra of SaeLM in Me2SO-d6 at 343 K. Expanded regions (δ 1H: 4.60–5.20 (A and B) and δ 1H: 4.60–5.20, δ 1H: 3.35–4.00 (C and D)) are shown. Glycosyl residues are labeled in roman numerals, and their carbons and protons are labeled with Arabic numerals

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