©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Analysis of the Mannan Region of Lipoarabinomannan from Mycobacterium bovis BCG
HETEROGENEITY IN PHOSPHORYLATION STATE (*)

Anne Venisse (§) , Michel Rivière , Joseph Vercauteren (1), Germain Puzo (¶)

From the (1)Laboratoire de Pharmacologie et de Toxicologie Fondamentales du CNRS, Département III, 31062 Toulouse Cedex and the Laboratoire de Pharmacognosie, 33076 Bordeaux Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lipoarabinomannan (LAM) is a major antigen of mycobacterial cell walls, involved in host-Mycobacterium interactions. In a previous work, LAM from the vaccine strain, Mycobacterium bovis BCG, was found to exhibit mannooligosaccharides at its arabinan nonreducing ends (ManLAM). The present report concerns the mannan core structure of this ManLAM. After partial hydrolysis of ManLAM, two populations of mannans (Ma1 and Ma2) were obtained by gel filtration chromatography. Their structural features were defined by means of two-dimensional homo- and heteronuclear (H-C) NMR sequences and methylation analysis. They were both found to be composed of an -(1 6)-linked mannan backbone with -(1 2)-Manp-linked side chains. They are highly branched, and Ma2 presents a higher frequency of branching than Ma1. Moreover, chemical analysis indicates that only Ma1 is phosphorylated. By a two-dimensional heteronuclear H-P total correlation experiment, the phosphate was found to be involved in a phosphodiester bond between inositol C-1 and glycerol C-3. Then, the molecular mass of mannan was established by mass spectrometry, which revealed a molecular mass of 3517 Da for the major molecular species of Ma1. Likewise, analysis of unfractionated mannans showed the occurrence of other, quantitatively minor molecular species, endowed with two phosphates.

This study clearly indicates that the mannan region of M. bovis BCG ManLAM exists as a heterogeneous population of molecules whose structures differ in their degree of glycosylation, level of branching, and phosphorylation state. The hypothesis that the relative abundance of these different molecules modulates the biological functions of LAM is discussed.


INTRODUCTION

Mycobacterium tuberculosis infects one-third of the world's population, and tuberculosis remains the largest cause of mortality in the world from a single infectious agent(1) . This emphasizes the need to understand the interaction of M. tuberculosis with host phagocytic cells and the immune system. However, little is known about the molecular basis of the pathogenesis of tuberculosis.

Lipoarabinomannan (LAM)()is considered as a major antigen of mycobacterial cell walls, widely distributed within Mycobacterium species(2) . LAM from M. tuberculosis has been demonstrated to exhibit a wide variety of biological activities such as suppression of T lymphocyte proliferation (3), inhibition of -interferon-mediated activation of murine macrophages(4) , and immunomodulation of a large array of macrophage cytokines(5, 6, 7) .

LAM from M. tuberculosis is composed of a mannan core linked to a linear arabinan chain to which oligoarabinosyl side chains are attached(8) . It was established that most of these side chains of the LAM from the virulent strain (Erdman) of M. tuberculosis are capped with either mono-, di-, or trimannosyl residues. This LAM is termed mannosylated and named ManLAM(9, 10) . This capping is missing from the LAM called AraLAM isolated from a rapidly growing strain of Mycobacterium, initially described as the avirulent strain of M. tuberculosis H37Ra(11) . Moreover, the reducing end of this AraLAM mannan core was found to be linked to a phosphatidyl-myo-inositol anchor (12, 13) similar to the mycobacterial phosphatidyl-myo-inositol mannoside (PIM) structure.

Some of the biological activities of LAM were shown to be related to characteristic structural features of these complex molecules. LAM treatment with mild alkali results in a decrease of its ability to suppress in vitro antigen-induced proliferation, -interferon activation of macrophages(4) , and cytokine release(6) . Acyl groups such as the fatty acids of the anchor but also the short fatty acids previously described (14, 15) seem to be crucial to these biological properties. The Man-capping at the LAM nonreducing termini also results in significant functional differences in vitro which could have important consequences in the pathogenicity of M. tuberculosis. AraLAM in vitro rapidly triggers tumor necrosis factor release from infected murine macrophages, thereby enhancing their bacteriostatic activity, whereas ManLAM from the virulent strain elicits low levels of tumor necrosis factor that would be too low to restrain the parasite's growth(5, 7, 16, 17) .

We previously reported the global structure of LAM from Mycobacterium bovis Bacille Calmette-Guérin (BCG) strain Pasteur, used throughout the world as a vaccine against tuberculosis(18) . We demonstrated that this LAM shares structural features with LAM from a virulent strain (H37Rv) of M. tuberculosis, since it is also capped by mannopyranosyl residues. Despite these data, ManLAM could still be considered as a virulence factor involved in mycobacterial invasion and/or survival in host cells. A more detailed structural definition of M. bovis BCG ManLAM was prevented by the considerable molecular heterogeneity of the fraction that was outlined during its molecular mass determination by matrix-assisted UV-laser desorption/ionization mass spectrometry. The present study was undertaken in order to define a more accurate structure of the mannan core of the ManLAM from M. bovis BCG and, more particularly, to establish the presence of a phosphatidyl-myo-inositol anchor at the reducing end of this molecule. Through the two-dimensional homo- and heteronuclear scalar coupling NMR and mass spectrometry analysis of the mannan, we unequivocally assess the presence of one phosphate group on an oligosaccharide of 20 glycosidic units. Moreover, the molecular heterogeneity of this fraction was shown to be size- and charge-dependent, especially by the occurrence of non-, mono-, and even diphosphorylated populations. Minor structural units causing this heterogeneity may be at the origin of important functional differences.

In view of the dependence of biological functions on structural difference, the implications of these structural observations in the role of ManLAM as a virulence factor will be discussed.


MATERIALS AND METHODS

Mild Acid Hydrolysis

The dManLAM from M. bovis BCG strain Pasteur was purified as described previously (18) and was treated with 0.1 N HCl, 100 °C, 15 min in order to specifically hydrolyze the arabinan chains. The hydrolysate was applied on a Bio-Gel P-4 or P-6 eluted with water, and samples were assayed by GC after hydrolysis and TMS derivatization.

Separation of Mannans by HPLC

Mannan fractions obtained after Bio-Gel P-6 were separated by anion exchange HPLC using an analytical Carbopac PA1 (Dionex) column at neutral pH. Elution was performed using a gradient of sodium acetate (ACS Grade, Merck) in deionized water (18 M) with a flow rate of 1 ml/min. Separation was monitored by pulsed amperometric detection (PAD II) (Dionex Corp.) after NaOH post-column addition. Using a two-split system, only 15% of the product was directed to the detector after addition of 300 µl min of NaOH solution (500 mM), and the remaining 85% were collected. HPLC was conducted on a Gilson (Gilson, France) gradient system.

Methylation

Mannans were O-methylated three times according to a modified (18) procedure of Ciucanu and Kerek(19) . Per-O-methylated products were hydrolyzed with 2 N trifluoroacetic acid at 110 °C for 3 h, reduced with NaBH, and peracetylated. The alditol acetates of methylated sugars were identified by GC/MS. Glycosidic linkage analysis was carried out according to Sweet et al.(20) .

Chemical Analysis

Quantitative carbohydrate determinations were routinely carried out by GC analysis after hydrolysis (2 N trifluoroacetic acid at 110 °C for 2 h) and TMS derivatization(18) . myo-Inositol content was measured following acid hydrolysis (6 N HCl, 110 °C, 18 h) and TMS derivatization. These conditions were proved to improve inositol recovery from inositol-1-P(21) . Glucitol was added as an internal standard prior to hydrolysis. Total phosphorus was determined after perchloric acid hydrolysis according the Bartlett procedure(22) .

GC and GC/MS

Routine gas chromatography (GC) was performed on alditol acetates and TMS derivatives of monosaccharides using a Girdel series 30 equipped with an OV-1 (0.3-µm film thickness, Spiral, Dijon, France) fused silica capillary column (25-m length 0.22-mm inside diameter). A temperature program from 100 to 280 °C at a speed of 3 °C/min was used.

GC/MS was performed on a Hewlett-Packard MS engine using both EI and CI ionization modes.

Mass Spectrometry LSIMS

LSIMS was performed on a ZAB 2E mass spectrometer (VG analytical). Spectra were generated by a 35-kV cesium ion beam with an acceleration voltage of 8 kV. Thioglycerol and (thioglycerol + 1% acetic acid) were used as matrix for negative and positive mode spectra, respectively.

NMR Spectroscopy

NMR spectra were recorded on a Bruker AMX-500 spectrometer equipped with an Aspect X32 computer. Samples were repeatedly lyophilized in DO for hydroxyl deuterium exchange and dissolved in DO (Spin et Techniques, Paris, France, 99.96% purity) at a concentration of 2 mg/ml in a 200 5 mm 535-PP NMR tube. All spectra were recorded at 303 K.

Power-gated H-decoupled phosphorus-31 (P) spectra (202 MHz) was recorded with 25-kHz spectral width, a 17-µs 90° pulse, and 3 s of relaxation time. The 16K acquired complex points were zero-filled to 64K and processed after exponential multiplication (line broadening = 5 Hz). Phosphoric acid (85%) was used as the external standard ( = 0.0).

The homonuclear Hartmann-Hahn (HOHAHA) (24) and rotating frame NOE (ROESY) (25, 26, 27) experiments were recorded without sample spinning using the standard pulse sequences supplied by Bruker, and data were acquired in the phase-sensitive mode using the time-proportional phase increment method(23) .

Heteronuclear correlation H {C} HMQC (28), H {C} HMBC (31) spectra were recorded in the proton-detected mode with a Bruker 5-mm H-broad band tunable probe with reversal geometry. The H {P} HMQC-HOHAHA was obtained according to Lerner's pulse sequence(30) , and a GARP sequence (29) was used for P decoupling during acquisition.


RESULTS

Chromatographic and Chemical Analysis

After an acid-catalyzed partial depolymerization of the deacylated molecule (dManLAM) we previously demonstrated that LAM from M. bovis BCG is mannosylated at its nonreducing ends(18) . The ManLAM hydrolysate (0.1 N HCl, 15 min, 100 °C) was applied to a Bio-Gel P4 column (data not shown) to separate the mono- and oligosaccharides from the nonhydrolyzed mannan (Ma). These mono- and oligosaccharides, arising from the arabinan domain, and the mannan core were identified from their monosaccharide composition routinely established by GC analysis after hydrolysis and TMS derivatization. The mannan core, eluted in the void volume, was predominantly composed of mannosyl units (Man) as expected and also of arabinosyl (Ara), inositol (Ins), and glycerol (Gro) units in small amounts. Moreover, P NMR analysis of Ma revealed one signal at 0.75 ppm, the chemical shift of which is not affected by pH in a range between pD = 6.8 and pD = 9.8, thus strongly suggesting the presence of a phosphodiester group.

The Ma core was analyzed by liquid secondary ion mass spectrometry (LSIMS) in both negative and positive modes (Fig. 1, A and B). In the high mass range, the negative mode-mass spectrum (Fig. 1A) is dominated by the peak at m/z 3037.9. Two other signals were observed at m/z 3015.9 and m/z 2999.8 distant from the base peak by 22 and 38 atomic mass units, respectively. These mass differences agree with the presence of sodium and potassium, suggesting that these three peaks characterize one molecular species. Thus, the m/z 3037.9 pseudomolecular ion contained one Na and one K leading to the following structure (M + Na + K - 3H). In order to support this ion assignment, Ma was analyzed in negative mode-LSIMS with a matrix doped with KI (data not shown). As expected, the peak at m/z 3037.9 was missing, and the mass spectrum was dominated by one signal at m/z 3015.9 assigned to (M + K - 2H). The positive mode mass spectrum of Ma (Fig. 1B) showed abundant cationized molecular ions (M + Na + K - H) at m/z 3039.6, supporting the previous interpretations. From all these data, the average molecular weight of M was established at 2978.4 Da. From this and from the Ma composition determined by GC analysis, we can propose that M, the major molecular species detected in Ma, was composed of 16 (Man, Ins), one Ara, one Gro, and two phosphates.


Figure 1: LSIMS analysis of Ma. A, negative mode LSIMS mass spectrum of Ma in a matrix of thioglycerol. The charge states are indicated on the figure for the major molecular species M. The second set of peaks downshifted from M by 162 atomic mass units (M`) presented a similar pattern and was interpreted in the same way. The ions at m/z 2676.8 and 2712.9 were assigned respectively to (M" + Na - 2H) and to (M" + Na + K - 3H) with M" = M - (2 162) atomic mass units. B, positive mode LSIMS spectrum of Ma in thioglycerol, 10% acetic acid. The M charge states are indicated. Two sets of peaks were observed, each dominated by ions at m/z 2877.5 and 2714.5 with a mass difference of -162 and -324 atomic mass units, respectively, from the base peak at m/z 3039.6. Two other sets of peaks appeared in a higher mass range and were not assigned.



Moreover, LSIMS analysis revealed two other sets of peaks down-shifted from M by 162 and 324 atomic mass units indicating the presence of two other molecular species, in lesser abundance, containing, respectively, one and two Man (or Ins) less than M. More interestingly, the high mass range of the positive mode-LSIMS spectrum shows two other sets of pseudomolecular ions at m/z 3561.5, 3539.4 and m/z 3399.3, 3377.2. The analogous ions were missing in the negative mode spectrum. Moreover, the mass difference between these ions and those assigned to M cannot be explained only by a different number of monosaccharide units.

In order to fractionate the various molecular species revealed by the LSIMS analysis, the ManLAM hydrolysate was chromatographed on a Bio-Gel P6 column monitored by refractory index (Fig. 2). Fractions 1 and 2, namely Ma1 and Ma2, respectively, were of interest since they predominantly contained mannosyl residues. The remaining oligosaccharidic fractions, 3 to 6, showed higher Ara/Man ratios and arose from the nonreducing termini of the arabinan domain(18) .


Figure 2: Bio-Gel P-6 chromatography of the dManLAM hydrolysate (0.1 N HCl, 15 min, 100 °C) eluted with water. Fractions 1 to 6 were analyzed by GC after hydrolysis and TMS derivatization. Samples 1 and 2 contained a high percentage of mannosyl residues (called Ma1 and Ma2), while samples 3-6 contained a higher Ara/Man ratio.



The molecular homogeneity of Ma1 and Ma2 was then checked by ion exchange chromatography with an analytical Carbopac PA1 column using an HPLC device monitored by an amperometric detector (Fig. 3). Gradient of sodium acetate in water was used as solvents, and sodium hydroxide was added post-column for amperometry detection. 80% of the Ma1 fraction was eluted with a retention time of 4.5 min (peak II) during the isocratic elution with water while most of the Ma2 appeared as a single peak (I) in the void volume (3 min). Small quantities of materials were detected with the sodium acetate gradient, between 15 and 20 min. The separation on Carbopac at neutral pH was dependent on the charge-to-mass ratio of the oligosaccharides. These chromatographic studies revealed the relative homogeneity of each fraction and suggested that fraction Ma1 was predominantly composed of a more strongly charged mannan population than Ma2.


Figure 3: Ion exchange HPLC profile (analytical column Carbopac PA1) of Ma1 (A) and Ma2 (B). The column was eluted for 10 min with water then with a gradient of 0.5 M sodium acetate in water for 20 min. The glycosidic samples were detected by amperometry as explained under ``Materials and Methods.'' In these conditions, a standard galactose sample was eluted in the void volume at 3 min while glucuronic acid and xylose-1-P were eluted in the gradient at 19 min and 25 min, respectively.



GC quantitative analysis established that Ma1 and Ma2 were composed by Aras and Mans in an approximate Ara/Man molar ratio of 1:20 and 2:20, respectively. In the same way, the ratio Ins/Man was estimated at 1:20 for Ma1 and 1:80 for Ma2. Finally, the phosphorus assay of each fraction indicated 0.7 mol of phosphorus/mol of Ma1 and 0.15 mol of phosphorus/mol of Ma2 (with an approximate molecular mass of 3000 Da for mannans).

Methylation data () obtained with Ma1 and Ma2 were in agreement with those previously reported for the ManLAM (18) where only mannopyranosyl and arabinofuranosyl ring forms were observed. Ma1 showed the presence of t-Manp and 2,6-linked Manp (40 and 35%, respectively) at twice the proportion of 6-linked Manp (16%). This latter unbranched Manp was found in lower proportions in Ma2 (9%), being 4-fold less abundant than 2,6-linked Manp (34%). Small amounts of t-Araf, 5-linked Araf, and 2-linked Manp residues were also present in both mannans. These data indicate that both mannans are highly branched.

Both fractions Ma1 and Ma2 were then analyzed by LSIMS, one-dimensional and two-dimensional homo- and heteronuclear H-C and H-P NMR.

LSIMS Analysis

The high mass range of the positive mode-LSIMS spectrum of Ma1 (Fig. 4A) showed an intense peak at m/z 3539.3. The presence of two other signals of lower intensity at m/z 3517.5 and m/z 3555.9 separated by -22 atomic mass units and +16 atomic mass units, respectively, from the base peak, suggested that the signal at m/z 3539.3 corresponds to the cationized molecular ion (M1 + Na). Thus, the proton and potassium-containing molecular ions were assigned to the peaks at m/z 3517.5 and m/z 3555.9, respectively. Moreover, these assignments were supported by the negative mode mass spectrum (Fig. 4B), showing an intense peak at m/z 3515.5 attributed to the pseudomolecular ion (M1 - H). From these values, the M1 average molecular mass was determined at 3517.0 Da. From the molecular mass of M1 and the chemical composition of Ma1, we can propose that M1 is composed of 18 Manps, 2 Arafs, 1 Ins, 1 Gro, and 1 phosphate group. A second set of ions was observed in lower abundance in both spectra and characterized another molecular species in Ma1 containing one Manp less.


Figure 4: LSIMS analysis of Ma1. A, positive mode LSIMS spectrum of Ma1 in a matrix of thioglycerol, 10% acetic acid. B, negative mode LSIMS spectrum of Ma1 in thioglycerol. Both spectra showed a second set of ions in lower abundance with a mass difference of -162 atomic mass units from the base peak.



Despite our efforts, the Ma2 LSIMS analysis was unsuccessful. Positive and negative mass spectra were devoid of pseudomolecular ions in the expected mass range. Nevertheless, the absence of Ma2 pseudomolecular ions can be explained by the absence of phosphate groups in this molecule.

We noted the absence, in the Ma1 and Ma2 LSIMS spectra, of the abundant pseudomolecular ion at m/z 3039.9 observed in the positive LSIMS spectrum of Ma and assigned to a mannan core with two phosphate groups.

In order to define the Ma1 and Ma2 structures more precisely, they were analyzed by one-dimensional and two-dimensional NMR spectroscopy.

NMR Studies of Ma1 and Ma2

The Ma1 and Ma2 proton anomeric resonance regions (Fig. 5) are dominated by two signals at 5.06 and 5.14 ppm (denoted B and D). In both spectra, the resonance at 5.14 ppm appeared to form a complex signal with two near-resonances at 5.11 (C) and 5.15 ppm. Two weaker superimposed signals at 4.915 and 4.93 ppm (denoted A) were also common to both mannans. However, they differed in their relative integration value, which was lower in the case of Ma2 (reported in Fig. 5). The downfield resonance at 5.20 ppm (F) was also observed in both spectra but was of lower intensity in Ma2. It can be noticed that a weak signal at 5.18 ppm (E) was only present in Ma2.


Figure 5: Expanded region of the one-dimensional H NMR spectra ( 4.3 to 5.2) of Ma1 (A) and Ma2 (B) anomeric protons are labeled A-F, as shown in Table I. The relative integration values are reported. Spectra were recorded over a spectral width of 5005 Hz using a 7-µs 90° pulse, 1.5 s of recycle delay and 1536 acquisitions of 1.63 s. Flame ionization detectors (16K) were zero-filled to 64K and multiplied by a exponential function (line broadening = 0.5 Hz) prior to Fourier transformation.



These anomeric protons and carbons were routinely assigned from the HMQC spectrum (not shown). The remaining protons and carbons of each spin system which characterize the units involved in Ma1 and Ma2 were partially attributed from the COSY (not shown), HOHAHA, HMQC, and HMBC spectra. The assignment of signals was also based on the chemical shifts of analogous compound NMR studies(18, 32, 33) . These values are summarized in Tables II and III.

Starting from the downfield anomeric resonances D at 5.14 and 5.15 ppm, the cross-peak in the HMQC spectrum showed a correlation with an upfield anomeric carbon resonance at 100.89 ppm. The C-1 chemical shift of an -D-Manp is described to be shifted upfield ( = 2 ppm) while its H-1 is deshielded ( = 0.2 ppm) upon 2-O-substitution(34) . Thus, both the anomeric carbon and the D protons were attributed to a 2-O-linked -Manp. This assignment was confirmed by the HMQC and HOHAHA spectra (Fig. 6) allowing the attribution of the C-2 resonance at 81.45 ppm and the H-2 resonances at 4.045 and 4.055 ppm. This downfield chemical shift of the C-2 resonance ( = 7 ppm) typified a 2-O-linked -Manp. Moreover, the C-5 resonance was localized at 74.05 ppm owing to the (C-5,H-1) correlation observed by HMBC (Fig. 7). This chemical shift is shifted upfield from its normal range ( = 2 ppm) () and is in agreement with glycosylation in C-6. Thus, this spin system was attributed to the 2,6-di-O-linked -Manp unit characterized by methylation analysis.


Figure 6: Partial two-dimensional HOHAHA spectrum ( 3.6-4.2/ 4.85-5.23) of Ma2. Magnetization transfer was performed by a 120 ms MLEV-17 sequence. Spectrum was recorded with a spectral width of 4500 Hz in both dimensions, a 4096 512 data matrix, and 16 scans for each t increment. The data matrices were zero-filled to 4K 1K points and multiplied by a /2 shifted squared sine-bell (SSB = 2) function in both dimensions before Fourier transformation.




Figure 7: Expanded zone of the H {C} HMBC spectra ( 4.9-5.06/ 68-83) of Ma1 and Ma2, showing connectivities involving the anomeric carbohydrate protons. Intra- and inter-residue correlations were identified. The intraresidue correlation between the H-1 of 6-O-linked Manp and the C-3 of this unit was only detected on the Ma1 spectrum (dotted line). Spectra were recorded in the magnitude mode with a spectral window of 4500 Hz in the F2 dimension (H) and 17.5 kHz in the F1 dimension (C) and 4096 512 time domain points with 20 (Ma1) or 32 (Ma2) scans per t increment and processed as described for the HOHAHA spectrum.



The remaining high intensity H-1 resonance B, at 5.06 ppm, correlated with the anomeric carbon at 104.9 ppm, was thus assigned to t--Manp since it was shown to be present in the same proportion as the 2,6-Manp by methylation analysis. This attribution was supported by the C-5 chemical shift of this spin system, found as previously on the HMBC spectra, at 76.12 ppm, 2 ppm downfield from a C-5 resonance of a 6-O-linked -Manp residue ().

The upfield superimposed anomeric proton resonances A at 4.915 and 4.93 ppm correlated on the HMQC spectrum with the weak signal C-1 at 102.16 ppm. These H-1 upfield shifts and the C-2 chemical shift resonance at 72.73 ppm were in agreement with a 6-O-linked -Manp. Moreover, the relative integration values of the anomeric protons (Fig. 5A): 9.3 for the H-1 B (t--Manp) and 4.7 for the H-1s A, in agreement with the methylation data (40% of t-Manp and 16% of 6-O-linked Manp) supported this attribution. From the HOHAHA spectrum, these H-1 resonances only showed connectivities up to H-4 resonance preventing assignment of C-5 and C-6 resonances. Nevertheless, since glycosylation on C-6 induces a downfield shift of this carbon signal of 4.5 ppm, it was assumed that the C-6 resonances of 6- and 2,6-O-linked Manp were superimposed at 68.35 ppm and were undistinguishable. Their H-6 shifts were then identified by HMQC but could not be discriminated.

The spin systems of the different monosaccharides identified by GC were all assigned except for the arabinosyl residues. By analogy with the NMR analysis of the ManLAM from M. bovis BCG, the resonance at 5.11 ppm called C was tentatively attributed to the H-1 of the -Araf. Unfortunately, this resonance did not show any correlation on HMQC spectra. A cross-section taken through this anomeric proton resonance showed connectivities at 4.16 ppm assigned to H-2 by COSY (not shown), with H-3 at 3.967 ppm and with H-4 at 4.07 ppm by HOHAHA. All these proton chemical shifts as well as the C-2 resonance established by HMQC at 82.9 ppm agree with an -Araf unit(35) .

From the significant anomeric proton F at 5.20 ppm, connectivity in the Ma1 HMQC spectrum was found with an anomeric carbon at 104.04 ppm. By means of the COSY and HOHAHA spectra, the protons were assigned only up to H-3. The corresponding carbon resonances were identified from the HMQC spectrum (see ) and, taken together, these values are consistent with an -Manp unit unsubstituted in positions 2 and 3.

The spin system E starting from the low intensity signal H-1 at 5.18 ppm showed only an H-2 correlation at 3.94 ppm in the Ma2 HOHAHA spectrum and was not identified.

As previously established from the methylation data, the NMR study confirmed that Ma2 contained a lower proportion of 6-O-linked -Manp units than Ma1. Moreover, the proton resonances H-1s attributed to 6-O-linked -Manp and to 2,6-di-O-linked -Manps both appeared as complex signals, suggesting a molecular heterogeneity of the mannan backbone.

The presence of phosphate in Ma1 and Ma2 and its location were investigated by NMR. Fig. 8represents the H-P HMQC-HOHAHA spectrum (A) and an expanded region of the HOHAHA spectrum (B) of Ma1. The one-dimensional P NMR spectrum shows a single resonance at -0.1 which is not affected by pH in the pD range from 6 to 10, in agreement with a phosphodiester group(36) . This phosphate was found to correlate, by H-P HMQC (not shown), with the following proton resonances: 3.92, 4.02, and 4.16. The H-P HMQC-HOHAHA sequence allows the magnetization transfer between P and all protons of the units linked to the phosphate group. The corresponding spectrum (Fig. 8A) shows connectivities with well defined proton resonances at 4.34, 4.16, and 3.4, and with overlapped proton resonances between 3.6 and 4 ppm. These proton resonances were assigned by means of COSY and HOHAHA experiments. The HOHAHA spectrum (Fig. 8B) reveals that the proton resonance at 4.34 correlated with five protons at 3.4, 3.62, 3.67, 4.16, and 4.34, whose multiplicity and chemical shift, reported in I, characterized the 1-phospho-myo-inositol protons(37, 38) . The most downfield signal at 4.34 which resonated independently on the one-dimensional H NMR spectrum as a broad singlet (6-7 Hz) was assigned to H-2, the only equatorial proton of the myo-Ins ring (unidentifiable triplet, J = J = 2.5 Hz). Likewise, the upfield triplet at 3.4 ppm (J = J = 9.2 Hz) characterized the H-5 of myo-Ins. The signal at 4.16 ppm resonated as a broad pseudotriplet with a coupling constant of 10 Hz and was found to correlate with phosphate in the H-P HMQC spectrum. From its chemical shift, multiplicity, and coupling constant values (J = 9.2 Hz, J = 9 Hz)(37, 38) , it can be put forward that this resonance typifies the H-1 of a myo-Ins whose C-1 is phosphorylated. The remaining proton resonances H-3, H-4, and H-6 were identified, respectively, at 3.62, 3.67, and 3.87 and agree with a myo-Ins ring (I). The H-P HMQC-HOHAHA spectrum showed other correlations with protons (between 3.7 and 3.76 and at 3.92 and 4.02) that did not belong to myo-Ins. It was described (36) that the H-2 and H-3,-3` proton resonances of a 3-phosphoglycerol overlap between 3.85 ppm and 3.95 ppm. Thus, the resonances at 3.92 and 4.02, which were found to correlate with the phosphate group by means of the H-P HMQC spectrum, were identified to the geminal glycerol protons H-3,-3` whose position was phosphorylated. On the H-P HMQC-HOHAHA spectrum (Fig. 8A), the remaining resonances between 3.7 and 3.76 were assigned ( 3.6 and 3.7), to the H-1,-1` of the 3-phosphoglycerol unit(36) .


Figure 8: A, H {P} HMQC-HOHAHA spectrum of Ma1 recorded at 500 MHz. B, the corresponding region of the Ma1 two-dimensional HOHAHA spectrum at 4.34 (H-2 Ins) showing the myo-inositol spin system. H {P} HMQC-HOHAHA spectrum was recorded with spectral windows of 4500 Hz in the F2 dimension (H) and 1000 Hz in the F1 dimension (P) and 4096 64 (time-proportional phase increment) point data matrix with 120 scans per t value. Relaxation delay was 1.5 s, and the mixing time 120 ms. The original data matrix point was expanded to a 4096 512 and processed as described for the HOHAHA spectrum.



The H-2 and H-6 of myo-Ins appeared to resonate 0.1 to 0.2 ppm downfield compared to the literature chemical shifts (37, 38, 39) (see I), suggesting that their carbons are substituted. Indeed, the expanded region of the two-dimensional rotating frame NOE (ROESY) spectrum of both mannans (Fig. 9, Ma2 ROESY spectrum) shows ROE contacts between the most downfield anomeric proton ( 5.20 ppm) attributed to an -Manp unit, not yet fully characterized, and the H-2 of myo-Ins. Thus, this correlation allowed the sequence -Manp(1 2)-Ins to be unambiguously established. The identical integration values found for this Man H-1 and the myo-Ins H-2 (Fig. 5) and H-5 resonances are in agreement with this interpretation. Unfortunately, ROE contacts were not observed starting from the H-6 resonance of myo-Ins. Nevertheless, its chemical shift is in agreement with a PIM-like structure with the main mannan chain linked to the myo-Ins C-6.


Figure 9: Partial two-dimensional H-H ROESY spectrum ( 3.6-4.4/ 4.85-5.23) of Ma2 showing connectivities involving the anomeric carbohydrate protons. Intra- and inter-residue correlations were identified. Experiment parameters are the same as those described in the HOHAHA except the mixing time which was set to 60 ms.



These NMR studies accurately established the structure of the Ma1 reducing end (Fig. 10). The phosphate group was bound to glycerol, via C-3, and to myo-Ins through C-1. The myo-Ins was found di-O-glycosylated, on C-2 and on C-6.


Figure 10: Structural models of mannan cores, Ma1 (A) and Ma2 (B), from M. bovis BCG dManLAM. These structural hypotheses take into account the molar ratios of the linked glycosyl residues and the molecular mass for Ma1. The exact distribution of side chains (regular or clustered), the position of the arabinan chain on the mannan cores, and the exact molecular mass of Ma2 remain to be determined.



Similar data were observed with Ma2. However, the relative integration of the myo-Ins H-2, on the Ma2 1D H spectrum with t-Manp H-1 intensity as reference, indicated that Ma2 contained 4 times less Ins than Ma1. Thus, in agreement with the Ma2 chemical composition (0.15 mol of phosphorus/mol of Ma2, Ins/Man = 1/80), it can be proposed that Ma2 was mainly composed (85%) of mannans devoid of the myo-inositol phosphoglycerol group at their reducing end. In the same way, for Ma1, if the relative integration value of Ins H-2 was fixed at 1, the total integration value for all the H-1s exceeded the unit number determined by mass spectrometry for the major molecular species by 20%. From this analysis and with the Ma1 phosphorus assay, it seems that Ma1 comprised 20% of molecules with neither Ins nor phosphate. All these data were also in complete agreement with the analytical ion exchange HPLC profiles (Fig. 3).

From our previous study on the dManLAM structure, it was assumed that the mannan core was composed by a backbone of (1 6)--Manp with side chains of (1 2)--Manp. Two-dimensional H NMR ROESY and H-C HMBC experiments performed on Ma1 and Ma2 supported these structures.

Indeed, the partial HMBC spectrum of Ma1 and Ma2 (Fig. 7) showed, beside the long range intraresidue correlations already described, connectivities between the H-1 of t-Manp (5.06 ppm) and the C-2 of 2,6-linked Manp (81.45 ppm). These data allowed us to unambiguously establish the interglycosidic linkage between the t-Manp and the 2,6-di-O-linked Manp. These mannans possessed a backbone of 6-O-linked Manp residues with 2-O-linked Manp units as side chains. Moreover, the Ma2 HMBC spectrum (Fig. 7) showed a correlation between the H-1 of 2,6-Manp at 5.14 ppm and the C-6 at 68.35 ppm, characteristic of a 6-O-linked mannopyranosyl unit. All the connectivities established by HMBC were confirmed by the ROESY spectrum (Fig. 9). ROE contacts were observed between H-1 of t-Manp (5.06 ppm) and H-2s (4.045 ppm, 4.055 ppm) and H-3 (3.945 ppm) of 2,6-linked Manp and between H-1 of 2,6-linked Manp (5.14 and 5.15) and H-2 (4.095 ppm), H-3, H-4, and H-5 of t-Manp. The carbons involved in the interglycosidic linkages of Ma1 and Ma2 are now clearly established; nevertheless, taking into account methylation and NMR data, a few sequences can be put forward for either mannan backbone as discussed below.

The mannans possessed a backbone of 6-O-linked Manp residues with 2-O-linked Manp units as side chains. The anomeric proton integration ratio of H-1 t-Manp/H-1 6-Manp was equal to 2 in Ma1 and reached 3 in Ma2, suggesting a higher proportion of side chains for Ma2, in agreement with the methylation data.


DISCUSSION

The basic structure of LAM, ubiquitously found in mycobacterial cell walls, consists of a mannan backbone to which an arabinan domain is attached(8) . Recently, LAM was subdivided into two types called ManLAM and AraLAM, according to the presence or absence of mannooligosaccharides capping the nonreducing ends of the arabinan side chains. The ManLAM was first found in the virulent M. tuberculosis strain Erdman(9) , while the AraLAM was described in a fast growing mycobacterial strain previously described as the avirulent M. tuberculosis strain H37Ra(11) . More recently, cell walls of M. bovis BCG, the vaccine strain, were also shown to contain ManLAM, which is not restricted to the virulent M. tuberculosis strains as postulated(18) . From this earlier study, using matrix-assisted laser desorption ionization mass spectrometry, the molecular mass of the ManLAM from M. bovis BCG was established at around 17.4 kDa. This analysis also revealed the considerable molecular heterogeneity that the ManLAM from a single strain displayed and which is estimated to be of 5 kDa. Similar features have been found for ManLAM from M. tuberculosis H37Rv. It was then considered that the variation in the number of glycosyl units among LAM molecules mainly accounts for the LAM heterogeneity.

It was also reported that structures of AraLAM from the rapidly growing strain of Mycobacterium and of its arabinose-free relative, lipomannan (LM) are based on the phosphatidylinositol mannoside (PIM) structure(13) . The reducing ends of their mannan regions are composed of a phosphatidyl unit. The most recent study involved LM hydrolysis with an exo- and then an endo--D-mannosidase, which yielded a Gro-P-Ins-Man unit similar to that from PIM. The presence of such a unit in AraLAM was first supported by GC analysis, after AraLAM hydrolysis, of Gro and Ins-1-P(12) . Then, per-O-alkyl oligoglycosyl alditols were produced from AraLAM and analyzed by analogy with those obtained from PIMs(9) . It was then proposed that a single -Manp unit was on C-2 of the Ins of AraLAM and the extended mannan emanated from C-6 of Ins.

In the present report, we wish to emphasize the mannan core structure from the M. bovis BCG ManLAM. As the arabinan is labile to mild acid, the mild hydrolysis of the ManLAM yielded the mannan core which was fractionated by gel permeation into two classes of mannan cores, namely, Ma1 and Ma2 according to their retention times. By anion exchange HPLC monitored by amperometry, the two mannan cores Ma1 and Ma2 appeared as relatively homogenous compounds. Interestingly, Ma1 was eluted with a higher retention time than Ma2. We observed a fortuitous separation of the mannans according to their charge by gel filtration. It was described that the Bio-Gel P-6 column presents an acidic character, such that when eluted with water, it tends to exclude negatively charged and retain neutral molecules(34) . The Ma1 and Ma2 structures were elucidated thanks to a novel analytical strategy involving the use of one- and two-dimensional homo- and heteronuclear NMR sequences and LSIMS. The structural models of Ma1 and Ma2 are shown in Fig. 10. They share a common structural feature assigned to a linear -(1 6) linked mannan backbone with single -(1 2)-Manp side chains. Beside this similarity, the two cores differ by their level of branching and by the presence or the absence of the phosphatidyl myo-Ins unit at their reducing end. So, the present paper demonstrates that mannan cores contribute to LAM heterogeneity, and, more interestingly, suggests that not all the LAM molecular species are endowed with a PIM structure.

The key analysis step was the identification of the phosphoric diester linkage at the reducing end of Ma1, thanks to a combination of two-dimensional homo- (HOHAHA) and heteronuclear H-P (HMQC-HOHAHA) NMR sequences. All these experiments were conducted on whole mannans obtained after alkaline and acid hydrolysis of LAM. Identical spectra were obtained with ManLAM after alkalinolysis. The two-dimensional H-P HMQC-HOHAHA analysis showed all the protons of the units linked to the phosphate. Among them, the most downfield signal at 4.34 was assigned to the myo-Ins H-2 through its multiplicity and chemical shift. Thus, this proton resonance was the starting point for the identification by HOHAHA of the remaining protons of the myo-Ins ring. H-1 resonated as a downfield triplet, indicating the linkage of C-1 to the phosphate group (J = 2.5 Hz; J = 9.2 Hz; J = 9 Hz). The other proton resonances of the HMQC H-P spectrum at 3.92 and 4.02 ppm were assigned to the geminal H-3, H-3` of the glycerol unit. Thus, from these long-range correlation experiments, phosphate was shown to be linked to myo-Ins and glycerol. Moreover, the downfield resonances of Ins H-2 and H-6 indicated that their positions are involved in a bond. Indeed, an inter-residue ROE connectivity was observed on the ROESY spectrum between the H-2 of the myo-Ins ( 4.34) and the H-1 F of a mannosyl unit ( 5.20), supporting the substitution of Ins C-2 with a mannosyl unit. Unfortunately, no similar ROE contact was observed from the H-6.

The most surprising feature of this report is the occurrence of nonphosphorylated molecules in the mannan fraction (Ma2), suggesting that a class of M. bovis BCG ManLAM is characterized by the absence of phosphatidyl-myo-inositol anchor. From literature, it could be hypothesized that this type of LAM arises from the arabinomannan (AM) found in the culture medium. Indeed, this neutral molecule from M. tuberculosis showed the same polysaccharidic structure as ManLAM, and its mannan core was briefly described as a linear mannan segment of -(1 6)-linked Manp with single -(1 2)-Manp as side chains(40) . However, ManLAM was extracted with ethanol from delipidated mycobacterial cell walls. Thus, with these extraction procedures, it can be expected that AM was absent from the ManLAM fraction.

Another important structural determination made in this report is the precise molecular mass of some mannans from M. bovis BCG by LSIMS. Such an analysis of Ma1 indicated the presence of a mannan with a molecular mass of 3517 Da. From the chemical composition and this value, it can be proposed for the first time that this mannan is composed of 18 mannosyl units, 2 arabinosyl residues, 1 inositol, 1 glycerol, and 1 phosphate group. Ma1 fraction also contained another mannan with one glycosidic unit less. LSIMS did not allow the molecular mass determination of molecules in Ma2, maybe due to their nonionic character. Moreover, LSIMS analysis of the mannan core mixture revealed molecular species containing two phosphates. Despite our efforts, these molecules were not observed in the LSIMS analysis of Ma1 or Ma2. The analysis of these fractions by anion exchange HPLC showed the presence, in small amounts, of compounds eluted in the salt gradient with a long retention time (between 15 and 20 min). It is well established that when mixtures are analyzed, LSIMS and FAB ionization techniques do not allow quantitative measurements. Thus, it can be proposed that quantitatively minor molecular species containing two phosphates produce abundant molecular ions. LAMs have already been described with two phosphates after a mild alkalinolysis of LAM(12, 14) , but the authors concluded that, beside the phosphatidyl-myo-inositol anchor, the second phosphate group occurred on the arabinan segment.

The structure of the mannan core appears to be complex, and a complete structural definition will require further experiments. The exact sequence of glycosyl units and the position of the arabinan chain on the mannan core remain to be determined. Assessment of the distribution of side chains will not be easy. Shoulders broaden the H-1 signals of 6-linked Manp and 2,6-linked Manp residues for both mannans on the one-dimensional H spectra. Subtle sequence changes can produce such effects on chemical shifts of anomeric protons, indicating that the mannan sequence might not be a regularly repeated one. However, each population could include molecules that differ very slightly, which could also explain these resonances. It is not often possible to distinguish variations among from variations within molecules. The mannan core represents only one-fifth of the entire molecule, and its diversity suggests how considerable ManLAM molecular heterogeneity is.

LAM is considered as a key molecule in macrophage-Mycobacterium interactions. Most of the recent data are based on the functional activity differences between ManLAM and AraLAM. The latter is a more potent inducer of tumor necrosis factor than ManLAM (5, 16, 17) and also increases the expression of early genes, involved in the activation of macrophages(7) . The failure of ManLAM to stimulate cytokine production and early gene expression could facilitate the virulent organism to multiply within the macrophages. All these functional differences were restricted to the presence or the absence of mannooligosaccharides at the nonreducing ends of the arabinan side chains of LAM. Nevertheless, it has been reported that most of the LAM properties are lost after LAM deacylation (4, 6). From the models proposed for the LAM structure, the acyl groups were limited to the myo-inositol phosphatidyl unit. However, other alkaline appendages like short acids (succinate and lactate) have been described in LAM from M. smegmatis and M. tuberculosis but never located(14, 15) . So, from the present results, it can be expected that only the ManLAM containing the phosphatidyl unit will be endowed with biological activities. Thus, the relative abundance of native ManLAM endowed or not with a phosphatidyl-myo-inositol unit could modulate the LAM functions. In order to assess this hypothesis, it becomes requisite to separate the different populations of native LAMs on the basis of their mannan structure and to define their biological properties. Identification of function-correlated structural features will enhance our understanding of LAM as a mycobacterial virulence factor. It would be of great interest to evaluate the ratio of these different populations among LAMs from mycobacterial species with different virulences.

  
Table: Methylation analysis of Ma1 and Ma2 from M. bovis BCG dManLAM


  
Table: Assignment of some protons and carbons of both Ma1 and Ma2 glycosidic units, based upon the interpretation of HMQC, HMBC, and HOHAHA spectra and data from literature. Chemical shifts are reported in parts per million and were measured in DO.


  
Table: Assignment of inositol protons based upon their chemical shift and multiplicity and data from literature



FOOTNOTES

*
This work was funded by grants from the Région Midi-Pyrénées RECH/9200841. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Association pour la Recherche contre le Cancer.

To whom correspondence and reprint requests should be addressed: Laboratoire de Pharmacologie et de Toxicologie Fondamentales du CNRS, Département III, 118 route de Narbonne, 31062 Toulouse Cedex Tel.: 33-61-33-59-12; Fax: 33-61-33-58-86.

The abbreviations used are: LAM, lipoarabinomannan; ManLAM, LAM with mannosyl units capping the arabinan ends; AraLAM, LAM with arabinofuranosyl termini; LM, lipomannan; PIMs, phosphatidylinositol mannosides; BCG, Bacillus Calmette Guérin; TMS, trimethylsilylation; HMBC, heteronuclear multiple bond connectivity spectroscopy; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; ROESY, rotating frame nuclear Overhauser spectroscopy; HPLC, high performance liquid chromatography; LSIMS, liquid secondary ion mass spectrometry; Ins, inositol; Gro, glycerol; t, terminal.


ACKNOWLEDGEMENTS

We thank Dr. M. Gheorghiu for the M. bovis BCG cultures and G. Tuffal for technical assistance in the use of pulsed amperometric detection.


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