(Received for publication, April 22, 1996, and in revised form, October 16, 1996)
From the Institut de Pharmacologie et de Biologie Structurale du Centre National de la Recherche Scientifique, 118 route de Narbonne, 31062 Toulouse Cedex, France
Lipoarabinomannans from fast growing
Mycobacterium sp., namely AraLAMs, stimulate the early
events of macrophage activation. The immunological activities of all of
these AraLAMs drastically decrease with the loss of the mild alkali
groups, which were believed to be restricted to the fatty acid residues
from the phosphatidyl-myo-inositol anchor. This report
reveals the presence and the structure of mild alkali-labile
phosphoinositide units linked via the phosphate to the C-5 of the
-D-Araf in the AraLAMs of
Mycobacterium smegmatis, a fast growing mycobacterial
species. Their structure was unambiguously established with a strategy
based on both one-dimensional 31P and two-dimensional
1H-31P heteronuclear multiple quantum
correlation spectroscopy (HMQC) and HMQC-homonuclear Hartmann-Hahn
spectroscopy NMR experiments applied to native AraLAMs and to AraLAMs
treated in mild alkali conditions. Next to these alkali-labile
phosphoinositides estimated at three per molecule, two other mild
alkali-stable phosphoinositide units were identified: the expected
(myo-inositol-1)-phosphate-(3-glycerol) unit typifying the
well known glycosylphosphatidylinositol anchor of the mannan core and,
more surprisingly, one
(myo-inositol-1)-phosphate-(5-
-D-Araf) unit having the same structure as the alkali-labile ones. Moreover, these four phosphoinositide units were found capping the arabinan side
chains. Thus, their different behavior toward mild alkaline hydrolysis
was explained according to their accessibility to the alkali reagent.
This novel class of LAMs, namely phosphoinositols-glyceroarabinomannans (PI-GAMs), are characterized by their phosphoinositide units but also
by the absence of fatty acid residues. These PI-GAMs were found to
elicit the secretion of tumor necrosis factor-
, suggesting that
phosphoinositides are the major PI-GAM epitope involved in this
process.
In recent years, the immunological findings concerning
lipoarabinomannans (LAMs)1 have indicated
that they play a key role in tuberculosis immunopathogenesis (1). These
lipopolysaccharide-like molecules are ubiquitously found in the cell
walls of the Mycobacterium genus (2). According to their
structure, LAMs have been classified into two types, the mannosylated
LAMs, called ManLAMs, and the AraLAMs. ManLAMs are characterized by the
presence of small (1-2)-manno-oligosaccharides located at the
arabinan side-chain termini. More recently, the ManLAMs have been
subdivided into two types according to the presence or absence of
phosphatidyl-myo-Ins anchor (3). ManLAMs were first isolated
from the Mycobacterium tuberculosis Erdman strain (4, 5) and
further from the vaccine strain Mycobacterium bovis BCG (6,
7). AraLAMs are devoid of manno-oligosaccharide caps, but the presence
of myo-Ins phosphate units capping a minor portion of the
AraLAM arabinan side chains was recently established (8). These AraLAMs
arise from an unidentified fast growing mycobacterial strain that was
initially considered as H37Ra (9).
AraLAMs mediate the early activation of macrophages. They stimulate
early gene responses, at the mRNA level, of the c-fos gene and chemotactic JE and KC cytokines (10, 11), but they also induce
transcription of the mRNA for cytokines characteristically produced
by macrophages (tumor necrosis factor- and interleukins 1-
,
1-
, 6, 8, and 10). The TNF-
transcription induced by AraLAMs was
blocked by monoclonal antibodies against CD14, an LPS receptor (12).
These AraLAM activities dramatically decrease when ManLAMs are used
(10), in agreement with the fact that virulent organisms survive and
multiply within the infected macrophages. However, ManLAMs mediate
other major specific interactions between immune cells and
mycobacteria. It was demonstrated that the ManLAMs from M. tuberculosis and M. bovis BCG bind selectively to
murine and human macrophages through the mannose receptor (13, 14),
suggesting that they mediate the binding of these mycobacteria to the
alveolar macrophages. More recent data (15) indicate that double
negative human CD4 CD8
T-cell lines
recognize ManLAMs from M. tuberculosis and
Mycobacterium leprae presented by human CD1b molecules.
All these ManLAM and AraLAM properties disappear after mild alkaline treatment, revealing the key role of the alkali-labile residues (16, 17). To date, from the ManLAM and AraLAM structural models proposed, these alkali residues are restricted to the fatty acids of the myo-Ins phosphatidyl anchor. However, Hunter and Brennan (18) reported the existence of alkali-stable and alkali-labile myo-Ins in the LAMs from H37Ra. The alkali-stable myo-Ins was assigned to the phosphatidyl-myo-Ins anchor located at the nonreducing end of the mannan core. Its structure was completely elucidated by two-dimensional NMR 1H-31P heteronuclear multiple quantum correlation spectroscopy-homonuclear Hartmann-Hahn (HMQC-HOHAHA) experiments, unambiguously establishing that the anchor was part of the LAMs and not due to possible phosphatidylinositol mannoside contamination (3). However, the precise location of the alkali-labile myo-Ins phosphate residues as well as their substituent structures were unknown (18).
Mycobacterium smegmatis is a fast growing mycobacterial
strain. A serologically active acidic arabinomannan was purified from M. smegmatis by Weber and Gray (19) and was fractioned into phosphorylated and nonphosphorylated molecules. Preliminary structural studies revealed that both forms have similar core structures characterized by 15-linked Araf residues attached to O-4
of Arap and 6-O-linked
-D-Manp residues. In the present work, we
have undertaken to explore the structure of the LAMs from M. smegmatis obtained by solvent cell wall extraction, using NMR
spectroscopy, and more particularly, we address the question of the
existence of alkali-stable and alkali-labile myo-Ins
phosphates, their substituent structures, and their locations.
The extraction and purification of M. smegmatis ATCC 607 AraLAMs were performed as described previously for the ManLAMs from M. bovis BCG (6). The M. smegmatis native LAMs monitored by SDS-polyacrylamide gel electrophoresis show a broad band located around 30 kDa, indicating that LAMs were not contaminated by lipomannans.
dAraLAMs were prepared from the crude fraction containing lipoglycan, namely LAMs and lipomannans (60 mg) treated with 0.1 N NaOH for 2 h at 40 °C followed by gel filtration on a Bio-Gel G75 column eluted by 0.1 N CH3COOH. The dLAM fractions were characterized by GC after hydrolysis and derivatization by a molar ratio of Ara to Man of 1.45:1.
Native AraLAMs (10 mg) were submitted to mild alkaline hydrolysis (0.1 N NaOD at 40 °C) in an NMR tube, and the reaction was monitored at different times (30 min, 1 h, 1 h 30 min, and 2 h) by 31P one-dimensional NMR spectroscopy. Then partial acidic hydrolysis of the dAraLAMs was performed with 0.06 N HCl for 15 min at 100 °C, and the hydrolysate after cooling was applied on a Bio-Gel P-4 column eluted by water. The collected samples were characterized by gas-liquid chromatography after hydrolysis (2 N trifluoroacetic acid for 2 h at 100 °C) and TMS derivatization.
Native AraLAMs (5 mg) were submitted in a NMR tube to partial acidic hydrolysis (0.1 N DCl for 15 min at 80 °C). The reaction was monitored by one-dimensional 31P NMR. The products of the reaction were then submitted to mild alkaline hydrolysis (0.1 N NaOD for 1 h at 40 °C) with no intermediate purification.
AraLAMs were O-methylated three times according to a modified (6) procedure of Ciucanu and Kerek (20). Per-O-methylated LAMs were hydrolyzed with 2 N trifluoroacetic acidic at 120 °C for 2 h, reduced with NaBD4, and acetylated. The alditol acetates were identified by GC/MS and quantified by GC. Table I reveals the presence per LAM molecule of the same ratio of t-Araf units and 3,5-di-O-linked Araf units.
|
Routine gas chromatography was performed on a Girdel series 30 chromatograph equipped with an OV1 wall-coated open tubular capillary column (25-m length × 0.22-mm inner diameter; Spiral, Dijon, France) using helium gas at a flow rate of 2.5 ml/min with a flame ionization detector at 310 °C. A temperature program from 100 to 280 °C at a speed of 3 °C/min was used. The injector temperature was 260 °C for TMS glycoside analysis. GC/MS analysis was performed on a Hewlett-Packard 5889X mass spectrometer (electron energy, 70 eV) working in both electron impact and chemical ionization modes, coupled with a Hewlett-Packard 5890 gas chromatograph series II fitted with an OV1 column (12 m × 0.30 mm).
NMR spectra were recorded on a Bruker AMX-500 spectrometer equipped
with an Aspect X32 computer. Samples were exchanged in D2O
(Spin et Techniques, Paris, France; 99.9% purity) with intermediate lyophilization and then dissolved in 99.96 atom % D2O at a
concentration of 40 mg/ml for the native LAMs and 6 mg/ml for the dLAMs
and analyzed in a 200 × 5-mm 535-PP NMR tubes. 1H
Chemical shifts are expressed in ppm downfield from the signal for
internal 3-(trimethylsilyl)-propane sulfonic acid sodium salt (H/TMS 0.015).
The one-dimensional proton (1H) spectrum was measured using a 30-90° tipping angle for the pulse and 1 s as a recycle delay between each of the 256 acquisitions of 1.64 s. The spectral width of 5005 Hz was collected in 16,000 complex points that were multiplied by an exponential function (LB = 2 Hz) 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 one-dimensional phosphorus (31P) spectra were measured
at 202 MHz by employing a spectral width of 25 kHz, and phosphoric acid
(85%) was used as the external standard (P 0.0). The
data were collected in 32,000 complex data sets, and an exponential transformation (LB = 5 Hz) was applied prior to processing to 64,000 real points in the frequency domain.
All two-dimensional NMR data sets were recorded at 35 °C without sample spinning, and data were acquired in the phase-sensitive mode using the time-proportional phase increment (TPPI) method (21).
The two-dimensional HOHAHA spectrum was recorded using a MLEV-17 mixing sequence of 30 ms (22). The spin-lock field strength corresponded to a 90° pulse width of 30 µs. The spectral width was 5000 Hz in each dimension. The HOD signal was presaturated for 1 s during the relaxation delay. 1024 spectra of 4096 data points with 16 scans/t1 increment were recorded.
1H-31P and 1H-13C correlation
spectra were recorded in the proton-detected mode with a Bruker 5-mm
1H broad band tunable probe with reversal geometry. The
single-bond correlation spectra (HMQC) of
1H-13C and 1H-31P were
obtained according Bax's pulse sequence (23). The GARP sequence (24)
at the carbon or phosphorus frequency was used as a composite pulse
decoupling during acquisition. For the 1H-13C
HMQC spectrum, spectral widths of 16,250 Hz in 13C and 4500 Hz in 1H dimensions were used to collect a 4096 × 400 (TPPI) point data matrix with 32 scans/t1 value
expanded to 4096 × 1024 by zero filling. The relaxation delay was
1.2 s. A sine bell window shifted by /2 was applied in both
dimensions. The 1H-31P HMQC spectrum was
obtained with a 4096 × 80 (TPPI) point data matrix with 64 scans/t1 value. The spectral window was 4003 Hz in F2 dimension (1H) and 810 Hz in F1 dimension
(31P). Relaxation delay was 1.2 s. The original data
matrix point was expanded to a 4096 × 512 real matrix. The pulse
sequence used for 1H-detected heteronuclear relayed spectra
(HMQC-HOHAHA) 1H-31P was that of Lerner and Bax
(25). These spectra were obtained with a 4096 × 80 (TPPI) point
data matrix with 64 scans/t1 value. The spectral
window was 4003 Hz in F2 dimension (1H) and 810 Hz in F1
dimension (31P). The relaxation delay was 1.2 s, and
different experiments were realized with different mixing times. The
original data matrix was expanded to a 4096 × 512 real
matrix.
THP-1 monocyte/macrophage human cell line was
maintained as nonadherent cells in continuous culture with Iscove's
modified Dulbecco's medium (Life Technologies, Inc.), 10% fetal calf
serum (Life Technologies) in an atmosphere of 5% CO2 at
37 °C. The purified molecules were added in duplicate to THP-1 cells
(0.5 × 106 cells/well) in 24-well tissue culture
plates and then incubated for 6 h at 37 °C. Stimuli were
previously incubated 1 h at 37 °C in the presence or absence of
10 µg/ml polymyxin B (Sigma) known to inhibit the
effect of LPS (26). Culture supernatants were then assayed for TNF
using the previously described cytotoxic assay against WEHI164 clone 13 cells (27). Each supernatant was tested at two dilutions (1:2 and 1:20)
in duplicate. The assay was performed as follows. 50 µl of sample
were added to 50 µl of WEHI cells (4 × 105
cells/ml) in flat-bottom 96-well plates. In each experiment, a
reference curve was obtained using serial dilutions of human recombinant TNF- (Life Technologies) starting at 5000 down to 1 pg/ml. After a 20-h incubation at 37 °C, 50 µl of tetrazolium salts (1 mg/ml 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide in Iscove's modified Dulbecco's medium,
Sigma) were added to each well and incubated for
4 h. Formazan crystals were solubilized with 100 µl of lysis
buffer (N,N-dimethyl formamide, 30% SDS (1:2) adjusted at pH 4.7 with acetic acid), and optical density was read at
570 nm with an enzyme-linked immunosorbent assay plate reader.
Fig.
1A presents the J-modulated
13C one-dimensional spectrum of the LAMs from M. smegmatis treated in mild alkaline conditions called dLAMs. This
spectrum shows the same anomeric carbon resonances as those present in
the partial DEPT 13C one-dimensional spectrum of the
M. bovis BCG dLAMs (Fig. 1B). However, they
differ by their relative intensity in the anomeric as well as in the
exocyclic aliphatic carbon regions (not shown). First, the resonances
at 103.42 ppm (V1) and 65.77 ppm
(V5) (Fig. 1A), assigned to the C-1
and C-5 of t--Arafs, are relatively more intense than
those of the analogous carbons of the M. bovis BCG dLAMs, so
the M. smegmatis LAMs contain relatively more
t-
-Araf units than the M. bovis BCG ones. The
second major difference is the decrease of the anomeric carbon
resonance intensity at
101.01 (VII1) (Fig.
1A) assigned to 2,6-di-O-linked
-Manps and maybe to 2-O-linked
-Manps, the latter of which are the ManLAM reporter
units. In order to clarify this assignment, the M. smegmatis dLAM 1H-13C HMQC spectrum was recorded (Fig.
2). The C-1 resonance at
101.01 (VII1) correlated with one anomeric proton
signal at 5.12 ppm, while in the M. bovis BCG dManLAM HMQC
spectrum (6), this C-1 resonance correlated with two H-1 signals at
5.25 and 5.22 ppm. These protons were assigned to the H-1s of
2,6-di-O-linked
-Manps and
2-O-linked Manps corresponding, respectively, to
the Manps from the mannan core and from the
manno-oligosaccharide caps. So these data unambiguously reveal the
absence of 2-O-linked Manp units in the LAMs of
M. smegmatis. Likewise, a single correlation was observed
between the M. smegmatis dLAM t-Manp C-1
resonance at 104.93 ppm (IV1) and the anomeric
proton at 5.06 ppm. In the case of the M. bovis BCG LAMs,
this anomeric carbon correlated with two H-1 signals assigned to the
t-Manps from the mannan core and the manno-oligosaccharide
caps. So, this result demonstrates that M. smegmatis LAMs
contain a single type of t-Manps restricted to the mannan
core. Thanks to the 1H-13C HMQC comparative
analysis, we propose that the M. smegmatis LAMs are devoid
of
-Manp-(1
[2)-
-Manp(1
]n
(0 < n < 2) oligosaccharides capping the
arabinan side chains and thus belong to the AraLAM class.
AraLAM Phosphorylation State
The native AraLAMs were analyzed
by one-dimensional 31P NMR spectroscopy (Fig.
3). The one-dimensional 31P spectrum shows
two distinct resonances at 0.11 ppm (P1) and
0.35 ppm (P2) with an
integration ratio P2:P1 of 4. The P2 and P1 chemical shifts, which are
not significantly affected by shifting the pD value from 6 to 10, were
assigned to phosphodiester groups (28). The P1 resonance was
tentatively assigned to the anchor phosphate based on the phosphate
chemical shift (
0.1 ppm) of the phosphatidyl-myo-Ins
anchor of M. bovis BCG ManLAMs (3). Moreover, from a
previous study the mild alkali-stability of this phosphodiester linkage
was established (3). Then, in order to support the P1 assignment, the
AraLAMs were submitted to mild alkaline hydrolysis (0.1 N
NaOD, 40 °C), and the reaction products were monitored by
one-dimensional 31P NMR spectroscopy. After 30 min of mild
alkaline hydrolysis (Fig. 4B), the P2:P1
ratio value decreases from 4 (Fig. 4A) to approximately 1 and remains constant with longer reaction times: 30 min (Fig. 4B) and 2 h (Fig. 4C). These data highlight the
presence of three mild alkali-labile phosphate units (P2) and two mild
alkali-stable phosphates (P1 and P2). One of the alkali-stable
phosphate, probably P1, can be assigned to the anchor phosphate, but
the presence of a supplementary and unexpected P2 alkali-stable
resonance makes this P1 assignment hazardous. So, to demonstrate the P1
attribution and also to establish the substituent structure of the
novel P2 alkali-stable unit, the AraLAMs treated in mild alkaline
conditions, namely dAraLAMs, were analyzed by two-dimensional
1H-31P HMQC (Fig. 5b)
and 1H-31P HMQC-HOHAHA (Fig. 5c)
experiments. The 1H-31P HMQC sequence allows
the assignment of the protons borne by the carbons involved in the
phosphodiester linkage, while the 1H-31P
HMQC-HOHAHA sequence leads to the definition of the substituent spin
systems.
Substituent Structure of the Two Mild Alkali-stable Phosphates, P1 and P2
The dAraLAM 1H-31P HMQC spectrum
(Fig. 5b) provides correlations between the P1 resonance and
three well-defined proton resonances at 4.16,
4.00, and
3.94. Since these protons resonate in an overcrowded area, their
assignments based on chemical shifts remain hazardous. To overcome this
problem, 1H-31P HMQC-HOHAHA spectra were
performed with different mixing times. Using a long mixing time (63 ms)
(Fig. 5c), the spectrum shows a complex set of correlations.
However, the cross-peak at
3.39 reveals a well resolved proton
resonance correlating on the HOHAHA spectrum (Fig. 5a) with
five protons whose chemical shifts and multiplicities agree with a
1-phospho-myo-Ins spin system (Table II). In
Table II, it can be seen that the myo-Ins H-1 (
4.16), H-2 (
4.35), and H-6 (
3.87) are downfield-shifted compared with
those of the 1-phospho-myo-Ins standard, in agreement with the glycosylation of the myo-Ins at the C-6 by the mannan
core and at the C-2 by one single
-D-Manp
unit. Moreover, the P1 linkage to the myo-Ins C-1 was
supported by the fact that H-1 (
4.16) resonates as a pseudodoublet
and as a pseudotriplet in the 1H-31P decoupled
HMQC-HOHAHA and in the HOHAHA spectra, respectively. The remaining
4.00 and
3.94 protons from the HMQC spectrum (Fig. 5b)
were assigned to the glycerol prochiral H-3 and H-3
, while H-1 and
H-1
are defined from the HMQC-HOHAHA spectrum (Fig. 5c) at
3.70 and 3.73 ppm. The weak intensity cross-peak observed at 3.96 ppm
on the HMQC-HOHAHA spectrum, using a short mixing time, reveals a
connectivity between P1 and Gro H-2. As expected, it is now clearly
established that the alkali-stable P1 corresponds to the phosphate unit
of the glycosylphosphatidylinositol anchor.
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The same NMR approach was applied in order to determine the substituent
structure of the unexpected alkali-stable P2 resonance. As for P1, the
HMQC spectrum shows that P2 correlates with three proton resonances at
4.14, 4.04, and 3.99 ppm (Fig. 5b). The HMQC-HOHAHA experiment reveals a complex pattern of connectivities. Again, the
proton at 3.34 ppm, which resonates independently in the
one-dimensional 1H spectrum, is attributed to H-5 from a
second myo-Ins spin system ( H-1 3.99;
H-2 4.27;
H-3 3.57;
H-4 3.67;
H-5 3.34;
H-6 3.77) (Fig.
5a). Likewise, P2 was found to esterify the second myo-Ins unit at the C-1 position from the multiplicity of
H-1 (t, J1,P = J1,6 9.7 Hz) in the HOHAHA spectrum (Fig. 5a). The H-1, H-2, and H-6
protons are upfield-shifted compared with those of P1
myo-Ins resonating at chemical shifts comparable with those of the 1-phospho-myo-Ins standard, establishing that
positions C-2 and C-6 are not glycosylated (Table II). Therefore, the
P2 myo-Ins unit is localized at the terminal position. The
remaining P2 proton connectivities (
4.14,
4.04) in the HMQC
spectrum (Fig. 5b) can be attributed to geminal protons from
an undetermined unit. From the 1H-31P
HMQC-HOHAHA spectrum (Fig. 5c) a cross-peak with a proton at 5.16 ppm is observed assigned to an anomeric resonance either of
2,6-di-O-linked
-Manp or of
t-
-Araf. The J1,2
coupling constant value of 3.15 Hz (Fig. 6) typifies a
-Araf unit (expected J1,2 of
-Manp and
-Araf of 1.8 and 4.1 Hz (29),
respectively). Therefore, the resonances at
4.14 and
4.04 were
attributed to the geminal H-5/H-5
protons of a
-Araf
unit, revealing that P2 esterifies this unit at the C-5. This C-5
phosphoester linkage is also supported by the downfield chemical shifts
of the H-5/H-5
(
H-5/H-5
-L-Araf
3.80/3.68 (30)). In the 1H-31P HMQC-HOHAHA
spectrum (Fig. 5c), the remaining cross-peak (
4.19) is
in agreement with the chemical shift of the H-2, H-3, and H-4
-Araf ring protons (
H-2 4.04, H-3 3.93, H-4 4.03)
(30). Also, the subspectrum at
0.35 (Fig. 6) reveals an extra
-Araf ring proton resonance superimposed with the H-5 at
4.04 ppm. Thus, it is proposed that the mild alkali-stable P2
esterifies a terminal myo-Ins at the C-1 position and a
-D-Araf at the C-5 position.
To support the structure of the
(-Araf-5)-P2-(1-t-myo-Ins) motif, the dAraLAMs
were hydrolyzed in mild acidic conditions (0.06 N HCl, 15 min, 100 °C). The reaction products were chromatographed on a
Bio-Gel P4 eluted with water and monitored for changes in refractive
index. The chromatogram (Fig. 7A) shows
mainly two peaks. The included sample (I) was hydrolyzed (2 N trifluoroacetic acid, 2 h, 100 °C) and was found
by GC analysis to be composed almost exclusively of Araf,
arising as expected from the arabinan moiety. The void volume
(E) was subdivided into five fractions (fractions 12-16).
By 31P one-dimensional NMR analysis, P1 was found in
fractions 13 and 14, both P1 and P2 in fraction 15, and P2 alone in
fraction 16 (Fig. 7B). This last fraction analyzed by GC
after acidic hydrolysis reveals Araf and myo-Ins.
Moreover, the two-dimensional HOHAHA experiment established that P2
esterifies a terminal myo-Ins unit (
H-1 3.99;
H-2
4.27;
H-3 3.57;
H-4 3.67;
H-5 3.34;
H-6 3.77) (not
shown). After peracetylation, fast atom bombardment-MS analysis in the
negative mode (not shown) demonstrated from the pseudomolecular ions
(M-H)
at m/z 727 that the major
molecular species is Ara-P2-myo-Ins. Other pseudomolecular
ions are observed in low abundance at m/z 943, 1159, 1375, and 1591, indicating that myo-Ins-P2 is also esterified by (Ara)n units, where n = 2-5,
respectively.
Taken together, these data allow us to propose the structure
(-Araf-5)-P2-(1-t-myo-Ins) for the mild
alkali-stable P2 phosphate and its localization at the nonreducing end
of the arabinan side chains.
In order to determine the substituent structures of
the alkali-labile P2 phosphates, the native AraLAMs were analyzed by
two-dimensional NMR spectroscopy as described previously for the
alkali-stable phosphates. As expected from the ratio value of P2:P1 of
4, the P1 cross-peaks in the 1H-31P HMQC-HOHAHA
spectrum (Fig. 8) are weaker than those of P2. However, from P1, the myo-Ins anchor can be characterized by the
proton resonances at H1 4.16,
H2 4.35,
H3 3.62,
H4
3.66,
H5 3.40, and
H6 3.88, while the glycerol is revealed by
the H-3 and H-3
(
4.00/3.94) proton resonances. Moreover, from the
Gro H-1, H-1
, and H-2 chemical shifts (3.70, 3.73, and 3.96 ppm, respectively), it can be advanced that the glycerol is not acylated (31, 32). This assignment was also supported by the absence of C-16 and
C-19 methyl fatty ester in routine GC analysis of the hydrolyzed
AraLAMs. From P2, this spectrum highlights the same connectivity
pattern as for the one observed from P2 resonance of the dAraLAM
1H-31P HMQC-HOHAHA spectrum (Fig.
5C), indicating that P2 is linked to
-Araf and
to t-myo-Ins. The t-myo-Ins is characterized by the cross-peaks at
H1 3.98,
H2 4.28,
H3 3.58,
H4 3.67,
H5 3.35,
H6 3.77, while the
-Araf is mainly
typified by the P2 connectivity with the H-1 at 5.16 ppm. So, from
these data, we propose that the three mild alkali-labile P2 phosphates
present the same substituent structures as the mild alkali-stable one, (
-Araf-5)-P2-(1-t-myo-Ins). All these
phosphoinositide units cap the arabinan side chains, and by analogy to
the ManLAMs, these LAMs were called
phosphoinositols-glyceroarabinomannans (PI-GAMs), since the
phosphatidyl anchor was found to be devoid of C-16 and C-19 fatty acids
(see Fig. 10).
To understand the different P2 behavior toward mild alkaline
hydrolysis, the following process monitored by 31P
one-dimensional NMR spectroscopy was applied to the PI-GAMs in
situ in the NMR tube. The PI-GAMs were successively hydrolyzed in
mild acidic (0.1 N DCl, 80 °C, 15 min) and mild alkaline
(0.1 N NaOD, 40 °C, 1 h) conditions. The
31P one-dimensional spectrum (Fig.
9A) of the acidic hydrolysis subproducts is
identical to that of the native PI-GAMs (Fig. 3). It is characterized
by two phosphodiester resonances P1 and P2 in a ratio approximately of
1:4. P2 arises from the (Ara)n-P2-t-myo-Ins motifs
(with n = 1-5), while P1 comes from the
myo-Ins-P-Gro unit of the mannan core. After alkaline
hydrolysis, the one-dimensional 31P spectrum (Fig.
9B) is dominated by one phosphodiester resonance assigned to
P1. The ratio P2:P1 is approximately 0.2, unambiguously indicating that
the P2 alkali-stable unit, observed when the PI-GAMs were treated in
mild alkaline conditions, was almost completely hydrolyzed from small
oligosaccharides (Ara)n-P2-t-myo-Ins. Thus, it can
be advanced that the P2 alkali stability is almost no more observed
when the P2 unit is contained by small oligosaccharides. Then it can be
proposed that the different behavior toward mild alkaline hydrolysis of
the (-Araf-5)-P2-(1-t-myo-Ins) motifs can be
explained in terms of PI-GAM conformation (Fig. 10)
that prevents the accessibility of the P2 alkali-stable unit to the alkaline reagents.
Induction of TNF-
The
potency of the PI-GAMs to stimulate the production of TNF- was
investigated using the human myelomonocytic cell line THP-1. Monocytes
were stimulated by various preparations of LAMs at 10 µg/ml
previously incubated in the presence or absence of polymyxin B (Fig.
11). PI-GAMs, like LPS, induced the THP-1 cells to
release TNF-
(250 and 550 pg/ml, respectively). In the presence of
polymyxin B, as expected, the LPS was found inactive, while the
induction of TNF-
by PI-GAMs was not significantly abrogated. However, the mild alkaline treatment of the PI-GAMs (0.1 N
NaOH, 40 °C, 2 h) decreases by a factor of 5 the production of
TNF-
. Surprisingly, the M. smegmatis LAM fraction devoid
of PI-GAMs was inactive in our bioassay even at 50 µg/ml. So, the
PI-GAMs appeared to be responsible for the TNF-
induction by the LAM fraction of fast growing mycobacterial species (33).
LAMs present a large spectrum of immunological activities
involving interactions with B-cells (1), phagocytes (16), and -T
lymphocytes (15). Some molecular targets of LAMs have either been
identified as or suggested to be the mannose receptor (13, 14), the
CD14 (12) receptor, and more recently the
T-cell receptor (15).
In order to define these molecular recognition mechanisms, precise
structural models must be proposed for the LAMs. Moreover, in order to
determine their role in the immunopathogenesis of mycobacterial
diseases, the precise knowledge of the LAM structures according to
their mycobacterial origins remains a major objective. Indeed, LAMs
that are ubiquitously found in the mycobacterial genus are involved in
specific immunological activities according to their mycobacterial
source. For example, AraLAMs from a fast growing mycobacterial species
present a higher capacity to induce TNF-
than ManLAMs from M. tuberculosis strain Erdman (34). Still more relevant is the fact
that ManLAMs from M. leprae and M. tuberculosis
activate specifically different
human T-cell lines
CD4
and CD8
(15).
From a structural point of view, whatever their mycobacterial origin,
the LAMs share the mannan core, the phosphatidyl-myo-Ins anchor, and the arabinan domain. Furthermore, according to the structures of the arabinan side chain extremity, the LAMs were subdivided into two types, AraLAMs and ManLAMs (4). The AraLAMs, characterized by -D-Araf caps (8), arise from
an unidentified fast growing mycobacterial species, while the ManLAMs
found in the slow growing species, M. tuberculosis (4, 5).
M. leprae (8) and M. bovis BCG (6, 7) are
typified by manno-oligosaccharides. However, the drawback of all the
structural and biological LAMs studies is the fact that LAMs are not a
single molecular species, as illustrated by the broad band observed in
SDS-polyacrylamide gel electrophoresis (35) and more recently by
matrix-assisted laser desorption-MS (3) studies showing precisely a
6-kDa molecular mass heterogeneity.
Nevertheless, it is now clearly established that all LAM activities, whatever their structure, require the alkali-labile residues. Thus, findings concerning the structure of LAMs, in their native forms, emphasize the importance of the development of new analytical approaches. Using two-dimensional 1H-13C and 1H-31P NMR experiments, we established that the LAMs from M. smegmatis, a fast growing mycobacterial strain, belong to the AraLAM class, but more interestingly the presence, the structure, and the location of mild alkali-labile and -stable phosphoinositides were unambiguously demonstrated.
First, the fact that the M. smegmatis LAM belongs to the
AraLAM class was established by one-dimensional 13C NMR and
two-dimensional 1H-13C HMQC experiments. From
the t--D-Manp and the 2-O-linked
-D-Manp anomeric carbon resonances, it was
found that each of them correlated with only one anomeric proton signal
instead of two in the case of the ManLAMs from M. bovis BCG
(6). Thus, the M. smegmatis LAMs are composed of
t-Manp and 2,6-di-O-linked Manp units
restricted to the mannan core, proving that they belong to the AraLAM
class. During the preparation of this paper, similar data appeared in the literature using a different analytical approach based on the
FAB-MS study of LAM acetolysates (8). However, this strategy, compared
with NMR analysis, requires LAM chemical degradation followed by
chromatography fractioning prior to FAB-MS analysis.
Second, unexpected structural features of mild alkali-labile and
alkali-stable phosphoinositides were deduced, for the first time, by
direct NMR studies of the native AraLAMs. The AraLAM one-dimensional
31P spectrum unambiguously reveals the presence of two
types of phosphodiester groups that resonate independently at 0.11
ppm (P1) and
0.35 ppm (P2) with an integration ratio of 1:4. Upon AraLAM treatment under mild alkaline conditions, P1 and P2 resonances are still present in the one-dimensional 31P spectrum, but
their relative intensities become similar, proving the presence of
three mild alkali-labile and two mild alkali-stable phosphodiester
groups per molecule (Fig. 10).
The structures of the P1 and P2 substituents were directly determined
from the native and the mild alkaline-treated AraLAMs by
two-dimensional 1H-31P HMQC-HOHAHA experiments.
Starting from the alkali-stable P1 resonance at
0.11, a diester
linkage with Gro and myo-Ins was established, typifying a
part of the well-known phosphatidyl-myo-Ins anchor.
Likewise, starting from the alkali-stable P2 resonance at
0.35 ppm,
the 1H-31P HMQC-HOHAHA spectra support a
phosphodiester linkage between a monosaccharide assigned to
-D-Araf (correlation with an anomeric proton,
J1,2 = 3.15 Hz) and myo-Ins. This NMR
approach applied to the native AraLAM P2 resonance at
0.35 ppm
reveals the same substituent structure
(myo-Ins)-P2-(
-D-Araf) for the
mild alkali-labile and mild alkali-stable P2. The terminal location of
the four P2-myo-Ins units was unambiguously determined from
their 1H chemical shifts. Moreover, the
1H-31P HMQC experiments from the alkali-stable
and -labile P2 show the same pattern characterized by three
connectivities, one with the H-1 of the t-myo-Ins and the
two others with the prochiral H-5 and H-5
of the
-D-Araf, revealing that P2 binds the
-D-Araf at C-5 and the t-myo-Ins
at C-1. On the basis of these NMR data, it was proposed that the
M. smegmatis AraLAMs contain two mild alkali-stable
phospho-myo-Ins, one assigned to the well known phosphatidyl-myo-Ins and the other to the unexpected
(t-myo-Ins-1)-P-(5-
-D-Araf), and
three alkali-labile units attributed to
(t-myo-Ins-1)-P-(5-
-D-Araf). All
four (t-myo-Ins-1)-P2-(5-
-D-Araf)
units were found to cap the arabinan side chains in a process involving
mild acidic AraLAM hydrolysis, P4 gel filtration, and oligosaccharide
analysis by 31P NMR spectroscopy and FAB-MS.
The different behavior of the four P2 motifs, having the same
substituent structures, toward mild alkaline hydrolysis was explained
in terms of accessibility precluded by the AraLAM conformation. It was
assumed that the three P2 alkali-labile units are exposed at the
molecule surface, while the P2 alkali-stable unit must be hidden in the
core of the molecule. This assumption was borne out by the fact that
the P2 alkali stability almost disappeared when the P2 units were
contained by small oligosaccharides
(t-myo-Ins-1)-P2-(5--D-Araf)-(
-D-Araf)n (n = 0-4) arising from AraLAM mild acidic
hydrolysis.
In conclusion, the structural study of the M. smegmatis
AraLAMs reveals the presence of four
(t-myo-Ins-1)-P-(5--D-Araf) units
capping the arabinan chains, three of these units being alkali-labile.
Myo-Ins-P-Araf motifs were also identified by
Khoo et al. (8) using FAB-MS analysis of
arabinose-containing oligosaccharides obtained either from LAM
acetolysates or enzymatically from alkali-deacylated LAMs produced by
an unidentified fast growing mycobacterial species. However, the
structure of these motifs was incomplete (the anomeric configuration
was not reported, and the sugar carbon involved in the linkage was only
suggested). Moreover, on the basis of our study, it can be suggested
that most of the
(t-myo-Ins-1)-P-(5-
-D-Araf) caps
were lost during the LAM deacylation prior to enzymatic degradation and
also during the permethylation step used to determine the proportion of
phospho-myo-Ins caps. Indeed, only a minor portion (around
20%) of the arabinan side chains were found to be capped by
P-myo-Ins in the case of the unidentified fast growing
mycobacterial species (8). The same approach applied to the M. smegmatis LAMs suggested the absence of such caps (8). Using the
same permethylation strategy (Table I), it was found that the number of
terminal
-D-Araf was equal to the number of
3,5-di-O-linked Arafs in agreement with the
absence of t-
-D-Araf caps. Nevertheless, from
Table I and from the molecular weight of the mannan
core,2 it can be advanced that 40-50% of
the arabinan side chains are capped by P2-myo-Ins units.
In the present report, we demonstrate that three phosphoinositide
motifs capping the arabinan side chains are alkali-labile groups.
Moreover, from the chemical shifts of the glycerol protons and
preliminary GC analysis, it was found that the AraLAMs arising from
M. smegmatis cell wall solvent extraction were devoid of C-16 and C-19 fatty acids, and are therefore probably localized at the
cell wall surface. This structural feature leads to two relevant
conclusions: (i) the definition of a new class of related AraLAM molecules, namely PI-GAMs (for
hospho
nositols-
lycero
rabino
annans), which are present in the crude lipoglycan fraction obtained from M. smegmatis cell walls, and (ii) the crucial importance of
the alkali-labile phosphoinositides that appear to be key structural motifs that support most of the specific immunological properties of
the lipoglycans concerning the stimulation of the macrophage activation
through CD14 (36), which is the major cell surface receptor for
monocyte activation.
Thus, the biological activity of the PI-GAMs in eliciting TNF-
secretion in a human monocytic cell line was compared with that of the
M. smegmatis LAMs devoid of PI-GAMs. It was clearly established that in the case of M. smegmatis strain, the
AraLAM activity concerning the induction of TNF-
is mainly mediated by the PI-GAMs. Indeed, the AraLAM fraction devoid of PI-GAMs and
characterized by the presence of C-16 and C-19 fatty acids (data not
shown) was found inactive, suggesting that the fatty acid residues of
the phosphatidyl myo-Ins are not involved in this process.
As described previously for the AraLAMs (10), the ability of the
PI-GAMs, treated in mild alkaline condition, to stimulate TNF-
production is drastically reduced, supporting the likelihood that
alkaline groups play a major role. Our data reveal that the exposed
phosphoinositides, which are alkali-labile, are of key importance and
may determine the macrophage early gene response during its interaction
with M. smegmatis. Thus, the ability of the PI-GAMs to
trigger TNF-
response in promoting a potent macrophage antimicrobial
activity could be the major molecular cause of the macrophage success
in killing M. smegmatis cells. Therefore, the PI-GAMs seem
important determinants of mycobacterial avirulence.
We gratefully acknowledge J. D. Bounéry for GC and GC/MS technical assistance.