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J Biol Chem, Vol. 275, Issue 1, 677-684, January 7, 2000
,From the Institut de Pharmacologie et de Biologie Structurale du Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse Cedex, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Mannosylated lipoarabinomannans are multifaceted
molecules. They have been shown to exert an immunosuppressive role in
the immunopathogenesis of tuberculosis. They are also described as antigens of host double negative The highly immunogenic lipoglycans ubiquitously found in the
mycobacterial envelope, the lipoarabinomannans
(LAMs)1 (1, 2), are thought
to play a major role in the immunopathogenesis of tuberculosis. They
regulate cytokine secretion (3-10), block the transcriptional
activation of interferon- It is now well established that LAMs, irrespective of their source, are
heterogeneous in size. This was first revealed by SDS-polyacrylamide
gel electrophoresis analysis where LAMs migrate as broad band around
30-40 kDa (15). The LAM molecular weight was more precisely determined
using matrix-assisted laser desorption ionization-time of flight mass
spectrometry. Indeed, it was found that LAMs are macromolecules of
around 17 kDa with a size distribution of at least 4 kDa (16). Thus,
any structural feature or biological parameter will be a weighted
average of the composite molecular species.
In an attempt to improve the purification of the Mycobacterium
bovis BCG LAMs, we developed a new extraction procedure leading to
two pools of ManLAMs, namely parietal and cellular (17). Structurally,
these ManLAMs differ mainly in the structure of the
phosphatidyl-myo-inositol anchor lipid moiety and the
percentage of manno-oligosaccharide caps (9). The parietal ManLAM
anchor corresponds to one acyl-form, characterized by acylation of the OH-1 of the glycerol residue by
12-O-(methoxypropanoyl)-12-hydroxystearic acid, a novel
fatty acid in the Mycobacterium genus, while the anchor of
the cellular ManLAMs exhibited a higher degree of acylation from a
combination of palmitic and tuberculostearic acids. In addition, these
two pools of ManLAMs were found to stimulate interleukin-8 and tumor
necrosis factor- We report here an elucidation of structural features of the ManLAMs
isolated from Mycobacterium tuberculosis H37Rv. Previous studies on the parietal LAMs (16) and on the total LAM fraction (18)
from M. tuberculosis H37Rv cells demonstrated that they belong to the ManLAMs class. The degree of mannose capping has been
estimated, from the total ManLAM fraction, to be around 40% (19).
The new extraction protocol developed for M. bovis BCG (9)
enabled isolation from M. tuberculosis H37Rv of two pools of LAMs, namely parietal and cellular. Their two major functional domains
were characterized as follows: (i) the cap motifs by capillary electrophoresis, (ii) the phosphatidyl-myo-inositol anchor
by NMR spectroscopy.
M. tuberculosis H37Rv ManLAMs Extraction and
Purification--
Parietal and cellular ManLAMs were purified as
described by Nigou et al. (9). Briefly, M. tuberculosis H37Rv cells were delipidated using
CHCl3/CH3OH, 1:1 (v/v). The delipidated
mycobacteria were extracted six times by refluxing in 50% ethanol at
65 °C for 8 h (parietal pool). The resulting cells (cellular
pool) were washed and disintegrated in ice by sonication and using a
French pressure cell as described previously (9). Each parietal and cellular extract was treated to remove proteins,
phosphatidyl-myo-inositol mannosides, DNA, RNA, and glucose
leading to glycanic- and lipoglycanic-rich extracts. Both extracts were
subjected to Triton X-114 phase separation. ManLAMs and LMs were then
separated by gel filtration, as described previously (9). The purified
ManLAMs and LMs monitored by SDS-polyacrylamide gel electrophoresis
were observed as broad bands around 35 and 20 kDa, respectively, as
described by Venisse et al. (16).
Acetolysis Procedure--
3 mg of ManLAMs were treated with 400 µl of anhydrous acetic acid/acetic anhydride, 3:2 (v/v), at 110 °C
for 12 h (20). The reaction mixture was dried and vortexed with
400 µl of cyclohexane/water, 1:1 (v/v). The cyclohexane phase which
contains the acylglycerol residues was analyzed by GC/MS, as
described previously (9).
GC and GC/MS Analysis--
GC was performed on a Girdel series
30 chromatograph equipped with an OV1 capillary column (0.22 mm × 25 m) using helium gas with a flow rate of 2.5 ml/min and a flame
ionization detector at 310 °C. The injector temperature was
260 °C and the temperature separation program was from 100 to
290 °C rising at 3 °C/min. GC/MS analysis were performed on a
Hewlett-Packard 5889X mass spectrometer (electron energy, 70 eV)
working in both electron impact and chemical ionization modes using
NH3 as reagent gas coupled with a Hewlett-Packard 5890 gas
chromatograph series II fitted with a similar OV1 column (0.30 mm × 12 m). Acetolysis products were analyzed on a 0.35-m length
column using a temperature separation program from 160 to 300 °C at
8 °C/min. The injector and interface temperatures were
290 °C.
Capillary Electrophoresis--
Analyses were performed on a
P/ACE capillary electrophoresis system (Beckman Instruments, Inc.) with
the cathode on the injection side and the anode on the detection side.
The electropherograms were acquired and stored on a Dell XPS P60
computer using the System Gold software package (Beckman Instruments,
Inc.).
Two micrograms of dried mild hydrolyzed (0.1 N HCl at
110 °C for 30 min) ManLAMs were mixed with 0.5 µl of 0.2 M 1-aminopyrene-3,6,8-trisulfonate (APTS) (eCAP
N-linked Oligosaccharides Profiling kit; Beckman) in 15%
acetic acid and 0.5 µl of a 1 M sodium cyanoborohydride solution dissolved in tetrahydrofuran (Aldrich) (21). The reaction was
90 min at 55 °C and the samples were then diluted in 9 µl of water
before injection. APTS derivatives were loaded by applying a 0.5 p.s.i. (3.45 kilopascal) vacuum for 5 s. The derivatives were
separated on a coated capillary column (eCAP N-CHO Coated Capillary
from eCAP N-linked Oligosaccharides Profiling kit; Beckman) of 50-µm internal diameter with 40-cm effective length (47 cm total
length). Analyses were carried out at a temperature of 20 °C with an
applied voltage of 24 kV using degassed Carbohydrate Separation Gel
buffer (eCAP N-linked Oligosaccharides Profiling kit;
Beckman) as running electrolyte. The detection system consisted of a
Beckman laser-induced fluorescence equipped with a 4-mW argon-ion laser
with the excitation wavelength of 488 nm and emission wavelength filter
of 520 nm.
NMR Spectroscopy--
Prior to NMR spectroscopic analysis, the
parietal (12.8 mg) and cellular (15 mg) ManLAM fractions were exchanged
in D2O (99.9% purity) at room temperature with
intermediate freeze-drying, and then dissolved in 400 µl of
Me2SO-d6 (99.8% purity, Eurisotop, Saint Aubin, France). The samples were analyzed in 200 × 5-mm 535-PP NMR tubes at 70 °C on a Bruker DMX-500 500 MHz NMR
spectrometer equipped with a double resonance (1H/X)-BBi
z-gradient probe head. Proton and carbon chemical shifts are expressed
in parts/million downfield from the methyl of
Me2SO-d6 (
Standards (1,2-diacyl-3-phospho-sn-glycerol,
sn-glycero-3-phospho-(1-D-myo-inositol),
and L- Using a new extraction method (17), two pools of LAMs, called
parietal and cellular, were isolated from the envelope of M. bovis BCG. This method was applied to the H37Rv cells (Fig. 1) and enabled recovery of 88 mg of
parietal and 135 mg of cellular LAMs. Their capping motifs and their
anchor domains were then characterized.
T-cells. Delimitation of
ManLAMs epitopes require knowledge of the precise structure of these
molecules. The two major functional domains (the cap motifs and the
phosphatidylinositol anchor) of the parietal and cellular ManLAMs of
Mycobacterium tuberculosis H37Rv were
investigated here. Using capillary electrophoresis, we established that
parietal and cellular ManLAMs share the same capping motifs, mono-,
di-, and trimannosyl units with the same relative abundance. By
31P NMR analysis of the native LAMs in
Me2SO-d6, the major acyl-form of
both parietal and cellular H37Rv ManLAM anchors, typified by the P3
phosphorus resonance, comprised a diacylglycerol unit. Three other
acyl-forms were characterized in the cellular ManLAMs. Comparative
analysis of the cellular Mycobacterium bovis BCG and M. tuberculosis ManLAM acyl-forms revealed the presence of
the same populations, but with different relative abundance. The
biological importance of the H37Rv ManLAM acyl-form characterization is
discussed, particularly concerning the molecular mechanisms of binding
of ManLAMs to the CD1 proteins involved in the presentation of ManLAMs to T-cell receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(10), and neutralize the potentially
cytotoxic oxygen free radicals (11). Mannosylated LAMs (ManLAMs)
selectively bind murine and human macrophages via the mannose receptor
(12, 13) and have been found to stimulate CD4/CD8 double negative and
CD8 T cells restricted by CD1 molecules (14).
secretion from human dendritic cells to different
extents (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
H/trimethylsilyl 2.52 and
C/trimethylsilyl 40.98). The one-dimensional phosphorus
(31P) spectra were measured at 202.46 MHz and phosphoric
acid (85%) was used as the external standard (P 0.0).
All two-dimensional NMR data sets were recorded without sample spinning
and data were acquired in the phase-sensitive mode using the
time-proportional phase increment method (22). Four two-dimensional
Homonuclear Hartmann-Hahn (HOHAHA) spectra were recorded using MLEV-17
mixing sequences of 9, 43, 82, and 113 ms (23). The
1H-13C and 1H-31P
single-bond correlation spectra (HMQC) were obtained using Bax's pulse
sequence (24). The GARP sequence (25) at the carbon or phosphorus
frequency was used as a composite pulse decoupling during acquisition.
The pulse sequence used for 1H-detected heteronuclear
relayed spectra (HMQC-HOHAHA) was that of Lerner and Bax (26).
-lysophosphatidylinositol) were purchased from
Sigma. L--Lysophosphatidylinositol, i.e.
monoacyl-sn-glycero-3-phospho-(1-D-myo-inositol) standard, was a mixture of approximately 82%
1-acyl-2-lyso-sn-glycero-3-phospho-(1-D-myo-inositol) (by integration of the 31P signal at 5.39 ppm obtained in
Me2SO-d6 at 343 K) and 18%
1-lyso-2-acyl-sn-glycero-3-phospho-(1-D-myo-inositol) (31P signal at 4.94 ppm).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 1.
Purification scheme for the parietal and
cellular ManLAMs and related compounds (AMs and LMs) from M. tuberculosis H37Rv.
Structure of the Manno-oligosaccharide Caps--
Parietal and
cellular M. bovis BCG LAMs were shown to belong to the
ManLAM class by two-dimensional 1H-13C HMQC
study on LAMs dissolved in D2O (17, 27). Indeed, the anomeric protons of t-Manp and 2-O-linked
Manp from the mannan core and the manno-oligosaccharide caps
resonate independently, corresponding to four independent spin systems.
However, an improved resolution in the 1H and
31P spectra was obtained by dissolving the multiacylated
ManLAMs (28) and LMs (29) of M. bovis BCG in
Me2SO-d6. H37Rv LAMs were then
analyzed in Me2SO-d6 by a complete
NMR strategy involving two-dimensional 1H-1H
COSY, HOHAHA with different mixing times,
1H-13C HMQC and 1H-13C
HMQC-HOHAHA in order to characterize the different spin systems which
compose the LAMs. This was conducted with help of the complete NMR
analysis of the mannan core of the parietal ManAMs carried out in
D2O (30) and the ManLAMs from M. bovis
BCG2 and of the data obtained
in Me2SO-d6 on the M. bovis BCG LMs (29). In this respect, 14 spin systems were
highlighted, assigned as 9 types of Araf (1 type of
3,5--Araf, 3 types of 5--Araf, 3 types of
2--Araf, and 2 types of -Araf) and 5 types
of Manp (2 types of t--Manp, 1 type
of 2--Manp, 1 type of 6--Manp, and 1 type
of 2, 6--Manp) (Table
I).
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Fig. 2 illustrates the anomeric expansion
area of the HMQC spectrum recorded in
Me2SO-d6 from H37Rv cellular LAMs.
The anomeric C-H pairs of t-Manp and
2-O-linked Manp were investigated to determine the H37Rv LAM class. We observed four anomeric C-H pairs, one at
99.2/4.90 (VIII1), one at 99.3/4.87 (VII1), and
two with the same 13C chemical shift and close
1H chemical shifts at 102.9/4.91 and 102.9/4.92
(IV1). They were assigned as the anomeric C-H pairs of
2,6--Manp (VIII), 2--Manp (VII), and
t--Manp (IV) from the manno-oligosaccharide
caps and the mannan core, respectively, by comparison to the NMR data
obtained on the M. bovis BCG LMs (29). Indeed, LMs are less
complex macromolecules as they are devoid of the arabinan domain. The
mannan core structure has been characterized by NMR leading to the
definition of the three main spin systems (2, 6--Manp,
t--Manp, and 6--Manp) in terms
of 1H and 13C chemical shifts (29). These three
spin systems were retrieved in the LAMs NMR spectra allowing
differentiation between the spin systems which belong to the mannan
core and those which correspond to the manno-oligosaccharide caps
present on the arabinan domain. Indeed, both
t-Manp proton spin systems were clearly observed on the HOHAHA spectrum (Fig. 3), one from
the H-1 at 4.91 and the other one from the H-1 at 4.92. In
addition, the two 1H spin systems of the
2-O-linked Manp (VII and VIII) assigned to
2--Manp and 2,6--Manp, respectively (Table
I), were conspicuous in the HOHAHA spectrum (Fig. 3). Similar results
were obtained for the parietal LAMs, demonstrating that parietal and
cellular H37Rv LAMs belong to the ManLAM class.
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The structures of these manno-oligosaccharide caps were then
investigated by capillary electrophoresis analysis as described previously (9). Both parietal and cellular ManLAMs were submitted to:
(i) mild acid hydrolysis, (ii) APTS tagging by reductive amination (21), and (iii) analysis by capillary electrophoresis monitored by
laser-induced fluorescence. The electropherogram from the parietal ManLAMs after 30 min hydrolysis is shown in Fig.
4. The peaks were assigned from APTS
standards and previous CE/ESI-MS studies concerning the structural
elucidation of the manno-oligosaccharide caps from M. bovis
BCG ManLAMs (9, 31). Peak I was assigned to free APTS reagent; peak II,
which corresponds to the major compound, was Ara-APTS; peak III,
Man-APTS; peak IV, Araf-Ara-APTS; peak V,
Manp-Ara-APTS; peak VI,
Manp-Manp-Ara-APTS; and finally peak VII,
Manp-Manp-Manp-Ara-APTS. The latter
compound was characterized by EC/ESI-MS analysis.3 The
extent of LAM hydrolysis was indicated by the relative intensities of peaks III and IV (Man-APTS and Araf-Ara-APTS,
respectively). The relative abundance of the different caps was
determined by integration of the peaks of interest (peaks V, VI, and
VII), revealing that the major structural motif was the dimannosyl unit
(66%), while the mannosyl and trimannosyl ones only represented 18 and 16%, respectively. Likewise, these manno-oligosaccharide cap
structures, with the same relative abundance, were characterized for
the cellular ManLAMs (not shown).
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Phosphatidyl-myo-Ins Anchor Acylation state--
The
phosphatidyl-myo-Ins anchor structure was investigated from
one-dimensional and two-dimensional phosphorus NMR. The one-dimensional 31P spectra of the parietal and cellular ManLAMs exhibited
broad unresolved signals in D2O (not shown) consistent with
multiacylated ManLAMs (9, 28, 29). No connectivities between phosphate and protons could therefore be obtained by two-dimensional
1H-31P HMQC and HMQC-HOHAHA. Recently, it was
shown by Nigou et al. (28) that
Me2SO-d6 is a suitable solvent for
recording high resolution one-dimensional 31P NMR spectra
of multiacylated ManLAMs. One-dimensional 31P spectra of
both H37Rv parietal (Fig. 5b)
and cellular (Fig. 5c) ManLAMs dissolved in
Me2SO-d6 were thus recorded. The
31P resonance were first assigned by comparison with the
chemical shifts of the 31P signals observed in the
one-dimensional 31P spectrum of the M. bovis BCG
cellular ManLAMs (Fig. 5a) (28). The one-dimensional
31P spectrum of the H37Rv parietal ManLAMs (Fig.
5b) showed one sharp resonance at 1.83 corresponding to
P3, while, for the cellular ManLAMs, four signals at 1.66, 1.72, 1.83, and 3.50 were observed assigned to P1/P2/P3/P5,
respectively (Fig. 5c). P1, P2, and P3 were
tentatively assigned to ManLAMs containing a diacyl-Gro residue, while
P5 corresponds to ManLAMs with a lyso-Gro residue. On the basis of the
one-dimensional 31P spectrum, it can be proposed that the
H37Rv parietal ManLAMs comprise one acyl-form typified by diacyl-Gro
residue, while at least three acyl-forms comprise the cellular ManLAMs.
In addition, the relative abundance of the acyl-forms of the H37Rv
cellular ManLAMs (Table III), determined from their signal heights, of
P1, 22%; P3, 67%; P4, 0%;, P5, 11%, differed from those observed
with the BCG ManLAMs (P1, 38%; P3, 42%; P4, 8%; P5, 12%). To
further elucidate the structures of these anchors, two-dimensional
1H-31P HMQC and HMQC-HOHAHA NMR experiments
were conducted. The 1H-31P HMQC-HOHAHA of
parietal ManLAMs (Fig. 6a)
exhibited one line of correlations for the P3 phosphate resonance. The
HOHAHA spectrum (not shown) can discriminate between the Gro and the
myo-Ins protons. From the H-5 at 3.10 ppm, the complete set
of correlations of the myo-Ins proton spin system was
assigned (H-1 4.02; H-2 4.11; H-3 3.24; H-4 3.45; H-6
3.59) from the coupling constants and chemical shifts. These chemical
shifts were consistent with the absence of fatty acyl appendage on the
myo-Ins. The Gro spin system defined from the
1H-31P HMQC spectrum (H-3/H-3' 3.86/3.80 ppm)
(Fig. 6b) and from the HOHAHA spectrum (H-1/H-1' at
4.36/4.14 ppm and H-2 at 5.12 ppm) corresponds to a diacylated Gro by
reference to the standard 1,2-diacyl-3-phospho-sn-glycerol unit in Me2SO-d6:
H-1/H-1' 4.33/4.13, H-2 5.10, H-3/H-3' 3.87/3.80.
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The 1H-31P HMQC-HOHAHA spectrum of cellular
ManLAMs (Fig. 7a) exhibited a
complex panel of correlations for P1, P3, and P5 phosphate resonance.
P1 and P3 showed correlations with downfield resonances at 5.12 and
5.09 in F2 dimension, which were assigned to
methine protons H-2 of diacylated glycerol units according to previous results (28, 29). A similar downfield correlation was not observed in
P5; instead the H-2 resonance is superimposed with the Gro H-3/H-3',
indicating lack of acylation of O-2 in this minor species (see below).
So, only P1 and P3 correspond to
1,2-diacyl-3-phospho-sn-glycerol units.
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From the H-2 Gro resonance, each glycerol spin system corresponding to
P1 and P3 was defined (Table II) from
inspection of the HOHAHA spectrum (Fig. 7d). Likewise, the
myo-Ins spin systems were identified (Table II) from the
correlations lines of both H-5 at 3.18 (P1) and 3.11 (P3) (in
F1 dimension) in the HOHAHA spectrum (Fig.
7c) in comparison with previous results (28, 29) and from
the multiplicity of the signals and the
3JHH coupling constants. The two
myo-Ins chemical shifts were relatively different. The P3
myo-Ins was assigned to a non-acylated
phospho-myo-Ins by comparison with a standard
(sn-glycero-3-phospho-(1-D-myo-inositol), H-1 3.65 (pd), H-2 3.88 (ps),
H-3 3.18 (pd), H-4 3.44 (t), H-5 2.99 (t), H-6 3.66 (t); pd,
pseudodoublet, ps, pseudosinglet, t, triplet). However, the P1
myo-Ins chemical shifts indicated that the
myo-Ins was acylated on C-3. This was apparent from the H-3
deshielding (
+1.36 ppm compared with the one in P3
myo-Ins ( 3.24)). We were unable, however, to attribute
the myo-Ins and Gro spin systems corresponding to P2 due to
the weak representation of this population.
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From these data, it can be proposed that P1 typified an anchor
comprising a diacylated Gro and a myo-Ins acylated on C-3, while P3 characterized an anchor formed by a diacylated Gro and a
non-acylated myo-Ins. Although the P5 signal was weak, we
were able to deduce the absence of an acyl residue on the C2 of the Gro, as no cross-peak was seen with a methine group around 5.10 ppm
(Fig. 7a). In comparison to a standard, a
L--lysophosphatidylinositol dissolved in
Me2SO-d6
(1-acyl- 2-lyso-sn-glycero-3-phospho-(1-D-myo-inositol), H-1/H-1' 3.99/ 3.97, H-2 3.75, H3/H3' 3.78/3.76), the Gro 1H chemical
shifts (Table II) indicated that the Gro was monoacylated on C1.
Moreover, from the chemical shifts of the myo-Ins protons (Table II), the absence of fatty acyl appendage on the
myo-Ins unit was deduced. P2 characterized an anchor
comprising a 1-acyl-2-lyso-Gro and a non-acylated
myo-Ins.
In summary, the major acyl-form of the parietal and cellular H37Rv
ManLAMs, typified by the P3 resonance, comprises a diacyl-Gro unit
(Fig. 8). In the case of cellular
ManLAMs, mono- and triacylated forms of the anchor were also
characterized. However, this NMR approach failed to identify a putative
fatty acid on the C-6 of the Manp unit linked to the C-2 of
the myo-Ins (32).
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In order to determine the nature of the acyl groups present on the
glycerol moiety, ManLAMs were submitted to acetolysis cleaving the
phosphate glycerol linkage, but preserving the acylglycerol residues
(20). These residues, extracted by cyclohexane/water partition, were
analyzed by GC/MS in electron impact and chemical ionization/NH3 ionization modes, as described previously
(9). The diacylated Gro was assigned to
1-tuberculostearoyl-2-palmitoyl-sn-Gro, while the lyso-Gro
forms correspond to both 1-palmitoyl-sn-Gro and
1-tuberculostearoyl-sn-Gro.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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To survive in its human reservoir, M. tuberculosis has evolved molecular strategies to control both innate and acquired immune responses. The survival of M. tuberculosis within the alveolar macrophages, a hostile environment, hinges on immunosupressive molecules. The infected host cells also sense the presence of mycobacteria from mycobacterial molecules that they recognize as foreign. This complex interplay between mycobacteria and hosts is mediated, on the one hand, by certain molecules, such those of the mycobacterial cell envelope, and on the other hand, by the activation of immune cells by the production of anti-microbial factors such as opsonins, peptides, cytokines, and chemokines.
Among the envelope molecules, LAMs are unique lipoglycans of the
Mycobacterium genus and are considered as the major
mycobacterial antigens (1). LAMs from pathogenic strains such as
M. tuberculosis, Mycobacterium leprae, and the
vaccine strain M. bovis BCG belong to the ManLAM class.
ManLAMs are multifaceted molecules. They have been shown to be involved
in the immunopathogenesis of tuberculosis as immunosuppressive
molecules (2). Moreover, they are described as antigens of host double
negative T-cells (33). These T-cells secrete antibacterial
peptides and interferon- and may be involved in protective immunity.
Delimitation of ManLAMs epitopes require knowledge of the precise
structure of the ManLAMs. To date, the ManLAMs from BCG, M. leprae, and M. tuberculosis share the same structural
model typified by the tripartite structure of a heteropolysaccharide of
D-mannan and D-arabinan, a
phosphatidyl-myo-inositol mannosyl anchor and the
Manp caps (1, 34). In addition, some decorative residues,
such as succinyl units, have been located on the arabinan domain (17).
It is now established that ManLAMs from any single source are
heterogeneous in size with respect to arabinan and mannan domains and
degree of acylation (1, 9, 28). Unfortunately, the marked heterogeneity
of ManLAMs hampers establishment of direct structure-function relationships.
Using the extraction procedure previously applied on M. bovis BCG (9, 17), the present report revealed the presence of two pools of ManLAMs, namely parietal and cellular, in the envelope of M. tuberculosis H37Rv. The location of these two pools of ManLAMs in the envelope of mycobacteria remains an open question. We surmised that the cellular ManLAMs were anchored in the plasma membrane as depicted by the envelope architecture proposed by Brennan et al. (35), while the parietal ManLAMs are incorporated in the outer layer with other amphipatic molecules as phosphatidyl-myo-inositol mannosides, LMs, and glycolipids, as depicted by the envelope model proposed by Rastogi et al. (36). Using this new extraction procedure, significant amounts of AMs and LMs were also found in both cellular and parietal extracts of M. tuberculosis H37Rv and M. bovis BCG envelopes. In summary, two pools of molecules (including LAMs, LMs, and AMs) were identified, which were extracted according to their location in the mycobacterial envelopes. However, it should be borne in mind the topology of these pools will probably be modulated by interactions with the mycobacterial environment.
A recent study on ManAMs demonstrated that parietal and cellular ManAMs share a structural model assigned to ManLAMs devoid of the phosphatidyl-myo-Ins anchor (30). Although these two molecules are structurally very similar, they have quite different immunological functions. These ManAMs, but not ManLAMs, have also been found in culture medium (37). It may be that ManAMs arise from ManLAMs after action of an endogenous mycobacterial glycosylphosphatidylinositol phospholipase-like as described for glycosylphosphatidylinositol-anchored proteins (38). However, the excreted ManAMs could also arise from the traffic of cellular ManAMs through the envelope.
The Manp caps seem to play a major role in ManLAM activity,
such as their binding to the mannose receptor (12, 13), and probably
also in the recognition of ManLAMs by the T cell receptor. By
capillary electrophoresis, we found that both M. tuberculosis H37Rv parietal and cellular ManLAMs share the same
cap structures assigned to Manp,
Manp1
2Manp, and
Manp12Manp12Manp units. The Manp12Manp motif corresponds to the
major one as in the ManLAMs from M. bovis BCG (9).
The structure of the phosphatidyl-myo-Ins anchors of M. tuberculosis H37Rv parietal and cellular ManLAMs was established using one-dimensional 31P and two-dimensional 1H-31P NMR. The same populations of anchors as those described for cellular M. bovis BCG ManLAMs (28) were found in cellular H37Rv ManLAMs, but with different relative abundance (Table III). In cellular BCG ManLAMs, the 31P resonance were divided into two groups according to their chemical shifts. The first group composed of P1, P2, and P3 (resonating around 1.8 ppm) corresponds to phosphates esterifying diacylated Gro units. The second group, which contains P4 and P5 (observed at lower fields around 3.5 ppm), corresponds to phosphates esterifying 1-acyl-2-lyso-Gro units. In the case of M. bovis BCG, cellular ManLAMs with diacyl-Gro units represent approximately 80%, and 38% (P1) being acylated on the C-3 of the myo-Ins (Table III). The remaining 20% (P4 + P5) of ManLAMs contain 1-acyl-2-lyso-Gro units, 8% (P4) being acylated on the C-3 of the myo-Ins. In the case of M. tuberculosis H37Rv, cellular ManLAMs with diacyl-Gro forms represent 89%, P3 being the most represented population (67%) and P1 being less abundant than in BCG (22%). For both species, parietal ManLAMs are composed by a single acyl-form, corresponding to P3 for H37Rv and typified by a 1-(12-O-(methoxypropanoyl)-12-hydroxy-stearoyl)-sn-glycerol for BCG (9). Taken together, these data indicate that the acyl-forms are more heterogeneous in the cellular than in the parietal ManLAMs. In summary, the major anchor structure of H37Rv ManLAM pool, typified by the P3 resonance, is at least a diacylated anchor (80%) (Fig. 8). In the case of BCG, the major ManLAM anchors, typified by P1 and P3 resonance, are at least tri- (35%) and di- (39%) acylated anchors.
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The fatty acyl appendage on the D-Manp linked to the C-2 of the myo-Ins residue described by Khoo et al. (32) could not be identified by the 31P NMR approach. We surmised that P1 typified a tetraacylated anchor (28), while the triacylated structure described by Khoo et al. (32) was represented by P2 or P3. Working on the total ManLAM pool, it is not possible to establish which acyl-form contains a fatty acid on the Manp. The definitive anchor structural determination with respect to the number of fatty acids will thus have to await purification of each acyl-form followed by mass spectrometric analyses.
Finally, M. tuberculosis ManLAMs have been found to
stimulate human double negative T-cells restricted by CD1b
molecules (39). Moreover, it has been established that the CD1
glycoproteins bind the ManLAMs through hydrophobic interactions via the
fatty acid residues of the anchor moiety (40). The molecular pathway can be subdivided in three main steps: (i) the ManLAM uptake by the
mannose receptor expressed on antigen presenting cell membranes, (ii)
the ManLAM binding to the hydrophobic CD1b groove, and (iii) the ManLAM
recognition by specific T cell receptors (33). Moreover, an
T-cell line (LDN4) was described proliferating in response to
M. leprae ManLAMs but did not respond to that from M. tuberculosis (14), suggesting ManLAMs structural differences.
However, to date, these ManLAMs share the same structural model.
Indeed, a precise structural ManLAM characterization is complex as
ManLAMs are composed of a large number of glyco- and acyl-forms.
Characterization of M. tuberculosis ManLAMs acyl-forms
should further understanding of the molecular mechanisms of ManLAM
binding to the CD1 proteins involved in the presentation of ManLAMs to
T-cell receptors.
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FOOTNOTES |
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* This work was supported by grants from the Région Midi-Pyrénées, pôle agroalimentaire (RECH/9702343), the Ministère de l'Education Nationale, de la Recherche et des Technologies (MENRT Microbiologie, 9710047).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-5-61-17-55-04; Fax: 33-5-61-17-59-94; E-mail: gilleron@ipbs.fr.
§ Present address: Div. of Membrane Biology, Central Drug Research Institute, Chhatarmanzil, PB 173, Lucknow 226 001, India.
¶ Present address: Unité 395-CHU Purpan, BP 3028, 31028 Toulouse Cedex, France.
2 M. Gilleron, G. Nigou, and G. Puzo, unpublished data.
3 B. Monsarrat, T. Brando, J. Nigou, M. Gilleron, and G. Puzo, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: LAMs, lipoarabinomannans; AMs, arabinomannans; BCG, bacillus Calmette Guérin; CD1, cluster of differentiation I; GC/MS, gas chromatography/mass spectrometry; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; ManAMs, AMs with mannosyl extensions; ManLAMs, LAMs with mannosyl extensions; LMs, lipomannans; 1H, 13C, and 31P NMR, proton, carbon, and phosphorus nuclear magnetic resonance; Manp, mannopyranosyl unit; Araf, arabinofuranosyl unit; myo-Ins: myo-inositol, Gro, glycerol; t, terminal.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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