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J. Biol. Chem., Vol. 280, Issue 31, 28347-28356, August 5, 2005

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

Kevin J. C. Gibson, Martine Gilleron¶, Patricia Constant¶, Bénédicte Sichi¶, Germain Puzo¶, Gurdyal S. Besra||, and Jérôme Nigou¶**

From the School of Bioscience, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom, the ^¶Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et de Biologie Structurale, CNRS, Unité Mixte de Recherche 5089, 205 Route de Narbonne, 31077 Toulouse cedex 4, France

Received for publication, May 19, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 (16)-Manp backbone is substituted at >80% of the O-2 position by side chains composed of Manp-(12)-Manp-(1. 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- (TNF-) 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 (16)-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Lipomannan (LM)¹ and lipoarabinomannan (LAM) are related powerful immunomodulatory lipoglycans found in mycobacterial cell walls (1–5). Their structures originate from a phosphatidyl-myo-inositol (MPI) anchor, which is mannosylated to generate LM (4, 6) 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 (12)-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). 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- and IL-12. For example, ManLAM are able to inhibit the LPS-induced production of IL-12 and TNF- (7, 8). 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 (8–10). 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) (11–13). This activity is likely to require PI caps, because AraLAM does not show any activity (14). Early studies demonstrated that LM from Mycobacterium sp. induce expression of pro-inflammatory cytokines (15). 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-, IL-8, and IL-12 (16–18). Furthermore, LM was shown to activate macrophages in a TLR-2-dependent, and TLR-4- and TLR-6-independent manner (16–18). 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). Induction of a protective pro-inflammatory response via TLR signaling should be to the benefit of the host (19–21), 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, 10).

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- and IL-12 (16, 22). However, the presence of the arabinan moiety on LAM inhibits the pro-inflammatory activity, presumably by masking the mannan core (23, 24) and thus limiting its accessibility to the TLR (16, 22). Also, the type of capping motifs may then direct LAM activity toward a pro-(PILAM) or anti-(ManLAM) inflammatory activity (3).

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, 26), Gordonia (27), Amycolatopsis (28), Corynebacterium (29), and most recently Turicella (30) and Tsukamurella (22). 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); however, upon chemical degradation of the arabinan domain, the resulting lipomannan moiety elicited a powerful pro-inflammatory response as previously demonstrated with ManLAM (16). Interestingly, the TpaLAM lipomannan moiety is composed of a linear (16)-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).

In the present study we report the isolation, structural, and functional characterization of a LM molecule from the Pseudonocardineae, Saccharothrix aerocolonigenes (31). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacteria and Growth Conditions—S. aerocolonigenes, type strain DSM 40034 (S. aerocolonigenes subsp. aerocolonigenes, recently renamed as Lechevaliera aerocolonigenes (32)) 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, 34). 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 -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 x 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 x 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). 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-x 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 _H 2.52 and _C 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 (_P 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) 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). Spectral widths of 25,154 Hz in ¹³C and 2200 Hz in ¹H dimensions were used to collect a 2,048 x 512 (time-proportional phase increment) point data matrix with 56 scans/t1 value expanded to 4,096 x 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by /2 was applied in both dimensions. A ¹H-¹³C HMBC spectrum was obtained using the Bax and Summers pulse sequence (38). Spectral widths of 25,154 Hz in ¹³C and 3,064 Hz in ¹H dimensions were used to collect a 2,048 x 480 (time-proportional phase increment) point data matrix with 80 scans/t1 value expanded to 4,096 x 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by /2 was applied in both dimensions. A ¹H-³¹P HMQC-HOHAHA spectrum was obtained using the Lerner and Bax pulse sequence (39). Spectral widths of 1,620 Hz in ³¹P and 3,064 Hz in ¹H dimensions were used to collect a 2,048 x 80 (time-proportional phase increment) point data matrix with 16 scans/t1 value expanded to 4,096 x 1,024 by zero filling. The relaxation delay was 1 s. A sine bell window shifted by /2 was applied in both dimensions. The 2D ¹H-¹H HOHAHA spectrum was recorded using a MLEV-17 mixing sequence of 110 ms (40). 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). The spectral width was 3,064 Hz in both dimensions. 512 spectra of 2,048 data points with 24 scans/t1 increment were recorded.

View larger version (29K): [in this window] [in a new window]   FIG. 1. SDS-PAGE analysis of S. aerocolonigenes lipoglycan. Lane 1, M. tuberculosis ManLAM and LM (top and bottom bands, respectively); lane 2, S. aerocolonigenes lipoglycan.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 5.03 (Ia₁), 4.98 (IIa₁), and 4.84 (IIIa₁), with nearly identical intensities (Fig. 2A). As revealed by the ¹H-¹³C HMQC spectrum, their corresponding anomeric carbons resonate at 101.4 (Ia₁), 102.4 (IIa₁), and 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, 43). The assignments are summarized in Table I. Spin systems Ia, IIa, and IIIa were unambiguously assigned to 2--Manp, t--Manp, and 2,6--Manp, respectively, in agreement with per-O-methylation data. The -anomeric configuration was demonstrated through the magnitude of their ¹J_H1,C1 coupling constants determined as 174, 171, and 169 Hz for spin systems Ia, IIa, and IIIa, respectively (-O-Me-Manp: ¹J_H-1,C-1 170 Hz; -O-Me-Manp: ¹J_H-1,C-1 161 Hz (44)). The pyranose ring was deduced from their _C-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, 43). Glycosylation at position 2 of 2--Manp (I) and 2,6--Manp (III) units was determined by the deshielding of their C-2 resonance at 77.0 and 77.8, respectively, as compared with the C-2 resonance of the unsubstituted t--Manp unit at 71.0 ( 6.0 and 6.8 ppm, respectively) (Fig. 2D and Table I). Glycosylation at position 6 of 2,6--Manp (III) units was shown through the deshielding of the C-6 resonance at 66.0 as compared with the C-6 resonance of the unsubstituted t--Manp unit at 62.4 ( 3.6 ppm) (Fig. 2D and Table I).

View this table: [in this window] [in a new window]   TABLE I Proton and carbon chemical shifts of SaeLM Chemical shifts were measured at 343 K in Me2SO-d6 and are referenced relative to internal Me2SO signals at H 2.52 and C 40.98.

View larger version (25K): [in this window] [in a new window]   FIG. 2. 1D 1H(A and B), 2D 1H-13C HMBC (C), and 2D 1H-13C HMQC (D and E) NMR spectra of SaeLM in Me2SO-d6 at 343 K. Expanded regions 1H: 4.60–5.20 (A), 1H: 3.35–4.00 (B), 1H: 4.60–5.20, 13C: 60–80 (C), 1H: 3.35–4.00, 13C: 60–80 (D), and 1H: 4.60–5.20, 13C: 97–105 (E) are shown. Glycosyl residues are labeled in roman numerals, and their carbons and protons are labeled with Arabic numerals. I, 2--Manp; II, t--Manp; III, 2,6--Manp; and IV, 6--Manp.

Spin systems Ib, -c, and -d characterized by _H-1 5.06, 5.08, and 5.11, respectively (Fig. 3A) and _C-1 101.4 (Fig. 2E) were attributed, by similarity to spin system Ia, as 2--Manp units (Table I). In a similar manner, spin systems IIb and -c, characterized by _H-1 4.96 and 4.92, respectively (Fig. 3A), and _C-1 102.5 and 102.8, respectively (Fig. 2E), were attributed to t--Manp units (Table I). Finally, spin systems IIIb and -c characterized by _H-1 4.86 and 4.87 (Fig. 3A) and _C-1 99.4 (Fig. 2E) were attributed to 2,6--Manp units (Table I). H-1 of the t--Manp unit (IIc₁) at 4.92 showed an intense inter-residue nOe contact with H-2 of 2--Manp unit (Id₂) at 3.90, whereas H-1 of these 2--Manp unit (Id₁) at 5.11 showed an intense inter-residue nOe contact with H-2 of 2,6--Manp unit (IIIb₂) at 3.79. As the main spin systems (Ia, IIa, and IIIa), these spin systems characterize the trisaccharide structure: Manp-(12)-Manp-(12)-[6)]-Manp-(1. The linkage of these units was further confirmed by HMBC experiment (Fig. 2A). Indeed, H-1 of the t--Manp unit (IIc₁) at 4.92 showed an inter-residue correlation with C-2 of 2--Manp unit (Id₂) at 77.8, whereas H-1 of these 2--Manp units (Id₁) at 5.11 showed an inter-residue correlation with C-2 of the 2,6--Manp unit (IIIb₂) at 78.6. In a similar manner, H-1 of spin system Ic (Ic₁) at 5.08 showed an intense inter-residue nOe contact with H-2 of spin system IIIc (IIIc₂) at 3.76 characterizing the sequence 2)-Manp-(12)-[6)]-Manp-(1. No obvious correlations could be observed with the remaining low abundant spin systems either in the ROESY or HMBC experiments. Thus one may conclude that the trisaccharide motifs characterized by these spin systems are probably located at a specific site in the structure, for example at the beginning or the end of the mannan chain.

MPI Anchor—The presence and structure of the MPI anchor was first investigated by 1D ³¹P and 2D ¹H-³¹P NMR of SaeLM dissolved in Me₂SO at 343 K. The 1D ³¹P showed four resonances at 1.83, 1.90, 3.58, and 3.83, in a ratio 1.0:1.0:1.4:1.8 indicative of the presence of different SaeLM acyl-forms (Fig. 4). As expected, in the 2D ¹H-³¹P HMQC-HOHAHA experiment, each phosphorus resonance correlated with a complex set of signals attributed to protons of myo-inositol and glycerol (Fig. 5, A and B). Protons H-1 of myo-inositol and H-3 and H-3' of glycerol were attributed using ¹H-³¹P HMQC experimentation (data not shown; Table I). The attribution of the remaining myo-inositol and glycerol signals was resolved using literature data to compare chemical shifts and multiplicity, and further confirmed by ¹H-¹H HOHAHA experiments (Fig. 5C and Table I) (43, 45). Glycerol units with phosphates resonating at 1.83 and 1.90 corresponded to diacylglycerol units, as revealed by the deshielding of their H-2 proton at 5.12 (Fig. 5A and Table I). In contrast, Glycerol units linked to phosphates resonating at 3.58 and 3.83 did not show a deshielded H-2 proton, corresponding to 1-acyl-2-lysoglycerol units (Fig. 5B and Table I). These attributions were in agreement with the chemical shift ranges of the corresponding phosphorus atoms, phosphodiacylglycerols (high field) versus phosphomonoacylglycerols (low field) (45, 46). The myo-inositol units derived from the four phosphorus atoms showed very similar proton chemical shifts that indicated non-acylated units (Fig. 5, A and B, and Table I). According to the nomenclature used in our previous studies (43, 45), the phosphorus atoms at 1.83 and 1.85 corresponded to P2 and P3, respectively, defining an MPI anchor characterized by a diacylglycerol and a non-acylated myo-inositol. The phosphorus atom at 3.58 corresponded to P5, whereas that at 3.83 corresponded to an acyl-form not previously characterized and termed P6. P6 actually seems to be the equivalent of P2, yet in this case the MPI anchor bears a mono-acylated glycerol. Thus, both P5 and P6 MPI anchors are characterized by a 1-acyl-2-lysoglycerol and a non-acylated myo-inositol. The difference between acyl-forms P2 and P3 on one hand and P5 and P6 on the other most probably arises from the presence or lack of an additional fatty acid positioned on the Manp unit linked at O-2 of the myo-inositol.²

View larger version (45K): [in this window] [in a new window]   FIG. 3. 1D 1H (A and B) and 2D 1H-1H HOHAHA m 110 ms (C) and ROESY m 300 ms (D) spectra of SaeLM in Me2SO-d6 at 343 K. Expanded regions ( 1H: 4.60–5.20 (A and B) and 1H: 4.60–5.20, 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. I, 2--Manp; II, t--Manp; III, 2,6--Manp; IV, 6--Manp; -Man-1, -Manp unit linked at O-6 of the myo-inositol; and -Man-2, -Manp unit linked at O-2 of the myo-inositol.

View larger version (20K): [in this window] [in a new window]   FIG. 4. 1D 31P spectrum of SaeLM in Me2SO-d6 at 343 K. An expanded region ( 31P: 0.0–6.0) is shown. *, this signal does not correspond to a phosphate from a MPI anchor and was not present in all investigations.

View larger version (41K): [in this window] [in a new window]   FIG. 5. 2D 1H-31P HMQC-HOHAHA m 47 ms (A and B), 2D 1H-1H HOHAHA m 110 ms (C), and ROESY m 300 ms (D) spectra of SaeLM in Me2SO-d6 at 343 K. Expanded regions 1H: 2.95–5.15, 31P: 1.55–2.15 (A), 1H: 2.95–5.15, 31P: 3.20–4.15 (B), 1H: 2.95–5.15, 1H: 3.02–3.16 (C), and 1H: 2.95–5.15, 1H: 5.11–5.18 (D) are shown. Numerals correspond to the proton number of the myo-inositol units, and numerals with letter G, to the proton number of the glycerol units. Protons H-2 of mannosyl units are labeled H2-M. -Man-1, -Manp unit linked at O-6 of the myo-inositol; -Man-2, -Manp unit linked at O-2 of the myo-inositol.

View larger version (33K): [in this window] [in a new window]   FIG. 6. Negative MALDI-TOF mass spectrum of SaeLM. 0.5 µlof a SaeLM solution at 10 µg/µl was mixed with 0.5 µl of the matrix solution (10 µg/µl of 2,5-dihydroxybenzoic acid in ethanol/water, 1:1, v/v) and analyzed by MALDI-TOF in the linear mode. The number on the top of each peak indicates the number of Manp units present on the corresponding main molecular species, i.e. tri-acylated.

TNF- Production by Macrophages—The potency of SaeLM to stimulate the production of TNF- was investigated using human THP-1 monocyte cell lines. SaeLM, when tested at concentrations of 10 and 20 µg/ml, induced a strong dose-dependent production of TNF- (Fig. 8A), which was not inhibited by polymyxin B (not shown), indicating that the observed cytokine induction was not due to LPS contamination. In contrast, mycobacterial ManLAM, known to be a poor inducer of pro-inflammatory cytokines, induced a very weak amount of TNF-.

It has been previously demonstrated that mycobacterial LM and PIM stimulate the production of TNF- in a TLR-2-dependent fashion (16, 48). To investigate the TLR dependence of TNF--inducing SaeLM activity, studies were conducted by measuring the inhibitory effect of cytokine production using specific anti-TLR-2 and anti-TLR-4 antibodies. As shown in Fig. 8B, whereas the anti-TLR-4 and an IgG2 isotype control antibodies had no affect on TNF- production induced by SaeLM, the anti-TLR-2 antibody inhibited this production. These data clearly underscore the role of TLR-2 in mediating the stimulation of TNF- production by THP-1 cells in response to SaeLM.

To gain better insights into the structure/function relationships, SaeLM activity was compared with that of M. bovis BCG LM (BCGLM) and TpaLAM lipomannan core (mahTpaLAM), both known to exhibit a pro-inflammatory activity. When tested at the same concentrations (10 and 20 µg/ml), SaeLM was found to exhibit a stronger TNF--inducing activity than BCGLM, the latter being more active than mahTpaLAM (Fig. 8A). These data confirm that the presence of an (16)-Manp chain is sufficient in providing pro-inflammatory activity for LM-like molecules as observed for mahTpaLAM. However, the presence of side chains, depending on their length and degree of sophistication, increases this activity. Nevertheless, one should not neglect the acylation pattern that might also influence the relative activity of these LM-like molecules,² because deacylated SaeLM (dSaeLM), obtained after an alkaline treatment, was unable to induce TNF- (Fig. 8A).

View larger version (11K): [in this window] [in a new window]   FIG. 7. Structural model of SaeLM. SaeLM contained an MPI anchor glycosylated by a (16)-Manp chain, which is further substituted, as indicated by integration of anomeric protons signals, at 83% (on average) of the O-2 positions by side chains composed of the Manp-(12)-Manp-(1 motif. MALDI/MS analysis showed that SaeLM was composed of a mixture of glyco-forms containing 19–61 Manp units. The distribution of glyco-forms was centered on the species containing 37 or 40 Manp units. The branched and linear portions of the (16)-Manp chain form distinct domains. The tri-acylated MPI anchor drawn, characterized by phosphate P3, corresponds to one of the SaeLM acyl-forms. m and n can be estimated on average at 11 and 2, respectively. The nature of the fatty acids (iC16:0, C16:0, and C18:1) is in agreement with the GC data; however, their relative distribution on the different acylation sites is unknown. M1, -Man-1; M2, -Man-2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this context, we report here the isolation and the structural and functional characterization of a new LM variant in the Pseudonocardineae, S. aerocolonigenes (31). The investigation revealed an original structure, and furthermore, we demonstrate that the LM possessed potent pro-inflammatory activity. SaeLM contained mannose as the sole carbohydrate. The main fatty acids esterifying SaeLM are 14-methylpentadecanoic, palmitic, and octadecenoic acids in agreement with the fatty acid composition found in the Saccharothrix genus (49). Per-O-methylation and detailed NMR studies revealed that SaeLM was composed from a (16)-Manp chain, which is further substituted at more than 80% of the O-2 positions by side chains composed of the Manp-(12)-Manp-(1 motif (Fig. 7). The structure of this mannan core, containing dimannoside side chains, is in contrast with that of mycobacterial LM where only single mannopyranosyl units substitute the (16)-Manp chain (4), except in a clinical isolate of M. kansasii where the LM has been reported to contain very few dimannoside side chains (50). Interestingly, some discrete trimannoside motifs, defined by spin systems Id, IIc, and IIIb, were identified and are probably located at a particular site in the structure, such as the beginning or the end of the chain. As previously stated the mannan domain of LM molecules is composed of a (16)-Manp chain substituted at some O-2 or O-3 positions by lateral side chains. On mycobacterial LM, one unanswered question remains: whether the linear and branched portions of the mannan core form distinct domains or whether 2,6--Manp and 6--Manp units intercalate at frequent regular intervals within the chain. In the case of SaeLM we found that proton H-1 of these two units correlated with their own C-6 resonances in the HMBC spectrum, and furthermore were distinguishable (Fig. 2C). Altogether, this suggests that 2,6--Manp on one hand, and 6--Manp units on the other hand, are interconnected and consequently form distinct domains.

1D ³¹P NMR analysis of SaeLM showed that the MPI anchor contained four acyl-forms, with a significant predominance of mono-acylglycerol (62%) as compared with di-acylglycerol (38%) acyl-forms. This distribution is in contrast with the data on M. tuberculosis or M. bovis BCG lipoglycans where the acyl-forms bearing diacylglycerols are the most abundant. In addition, position 3 of the myo-inositol was not acylated. Acylation of myo-inositol on lipoglycans actually seems to be restricted to the Mycobacterium genus, because it was not observed in any of the lipoglycans investigated so far arising from the genera, Rhodococcus (25, 26), Tsukamurella (22), Amycolatopsis (28), or Turicella (30). These findings are surprising because acylation of myo-inositol was, at least, observed on PIM, in Rhodococcus equi (25). This suggests that there is a different fatty acid modeling system in mycobacteria compared with the other actinomycetes genera. However it has been shown that mycobacterial lipoglycan biosynthesis occurs mainly from tri-acylated PIM precursors (6, 51, 52). Moreover, position O-3 of myo-inositol is the last one to be acylated during biosynthesis (48, 53), and as such, tetra-acylated forms of PIMs may constitute a storage pool of PIMs. MPI anchors corresponding to P2 and P3 on one hand and P5 and P6 on the other hand do not show, as revealed by 2D ¹H-³¹P HMQC-HOHAHA, any difference in terms of acylation of the myo-inositol and the glycerol units. The difference probably arises from the acylation state of the fourth potential acylation site of the MPI anchor that cannot be investigated by this approach, i.e. position 6 of the Manp unit linked at O-2 of the myo-inositol (1, 4). Indeed it has been found, after purification of the individual acyl-forms of M. bovis BCG LM and analysis by MALDI-MS that P2 and P3 MPI differ by the presence of an additional fatty acid, with P3 containing the additional fatty acid.² In a similar way, P6 defined in the present study, which seems actually to be the equivalent of P2, is likely to differ from P5 by the absence of a fatty acid on the Manp unit, because P5 has been shown to bear one in this position.² SaeLM MPI anchor was found to be based on a diglycosylated myo-inositol unit substituted at positions O-2 and O-6, by a t-Manp unit and the mannan core, respectively. Indeed the Manp units glycosylating the myo-inositol were clearly identified by NMR experiments. H-1 of -Manp-1 (linked at O-6 of myo-inositol) and of -Manp-2 (linked at O-2 of myo-inositol) units were deshielded as compared with the other carbohydrate anomeric resonances and consequently could be detected despite their low abundance. They were clearly identified on the ROESY spectrum due to intense nOe contacts between their H-1 and H-2 or H-6 of myo-inositol, respectively (Fig. 5). The different anomeric protons of -Manp-2 units corresponding to the acyl-forms characterized by the phosphates P2, P3, P5, and P6 could be readily distinguished. However the anomeric protons of the different -Manp-1 units may overlap.

View larger version (20K): [in this window] [in a new window]   FIG. 8. TNF- production by human THP-1 monocyte/macrophage cell line in response to SaeLM and various stimuli. A, ManLAM, SaeLM, BCGLM, mahTpaLAM, or dSaeLM were tested at 10 (black bars) and 20 (white bars) µg/ml. Polymixin B, when previously added, had no effect on the amount of TNF- released by these stimuli (not shown). LPS from E. coli 055:B5, at a concentration of 0.2 µg/ml, induced 1020 pg/ml TNF-. B, TLR dependence of SaeLM pro-inflammatory activity. SaeLM at 10 µg/ml and the various antibodies (anti-TLR-2, anti-TLR-4, and IgG2 isotype control) at a concentration of 10 (black bars) or 20 (white bars) µg/ml were added to THP-1 cells. LPS at a concentration of 0.2 µg/ml induced 510 pg/ml TNF-. ManLAM and LM were from M. bovis BCG; mahTpaLAM was prepared as previously described (22). BCGLM, LM from M. bovis BCG; dSaeLM, deacylated SaeLM; mahTpaLAM, mild acidic hydrolyzed TpaLAM, i.e. TpaLAM lipomannan core.

In summary, SaeLM exhibits an original structure with the same core domains as described for mycobacterial LM, but with some differences in acylation, branching, and size of the side chains present on the mannan core. Altogether SaeLM, with dimannoside side chains, appears to be the most elaborated non-mycobacterial LM molecule identified to date (Fig. 7).

LM (16–18) and lipomannan-domain-containing molecules (22) possess conserved structural motifs that are able to induce pro-inflammatory activity, however, in order for optimal induction, this structural moiety must be readily accessible to the host receptor(s). Interestingly, TpaLAM lipomannan core (mahTpaLAM), composed of an (16)-Manp chain with no side chains, is a powerful inducer of TNF-, demonstrating that an (16)-Manp chain is sufficient in providing pro-inflammatory activity and that branched t-Manp units are not necessarily required. LM-like molecules activate immune cell responses via TLR-2; however, the molecular dynamics of LM-like molecules binding to TLR-2, in particular, and in general of TLR ligands binding to their receptor(s) remains unclear (54). Accessory molecules such as LBP or CD14 (16) or heterodimerization with another TLR such as TLR-1 (55) participate in the recognition process of LM. Nevertheless, a direct interaction between the pathogen-associated molecular patterns and TLR-2 receptor has been clearly shown to be involved in signaling (56). Here we found that SaeLM exhibited a stronger TNF--inducing activity than M. bovis BCG LM, the latter being more active than mahTpaLAM. Altogether these data establish that a linear (16)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity. However adding side chains and extending their size increases this activity, most probably as a result of a higher affinity for TLR-2. In addition, the acylation pattern might also influence the relative activity of these LM-like molecules, because deacylated SaeLM (dSaeLM), obtained after an alkaline treatment, was unable to induce TNF-. Altogether, our findings provide better understanding of the structure/function relationship of TLR-2 lipoglycan-dependent signaling. In addition, and of more general interest, this study participates in the effort of deciphering the molecular basis of the recognition of pathogen-associated molecular patterns by TLRs.

	FOOTNOTES

 
^* This work was supported in part by grants from the CNRS (France) (to G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Servier Laboratories Ltd., Gallions, Wexham Springs, Wexham, Slough SL3 6RJ, UK.

^|| Supported as a Lister Institute-Jenner Research Fellow and by grants from the Medical Research Council (UK) (Grant G0200510) and the Wellcome Trust (Grant 058972).

^** To whom correspondence should be addressed: Tel: 33-561-175554; Fax: 33-561-175994; E-mail: jerome.nigou{at}ipbs.fr.

¹ The 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-, tumor necrosis factor ; TpaLAM, T. paurometabola lipoarabinomannan; BCG, bacillus Calmette-Guerin; nOe, nuclear Overhauser effect.

² M. Gilleron, J. Nigou, D. Nicolle, V. Quesniaux, and G. Puzo, submitted manuscript.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Thérèse Brando (Institut de Pharmacologie et de Biologie Structurale, Toulouse) for expert technical assistance with capillary electrophoresis/laser-induced fluorescence experiments.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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