From the
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 (
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.
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)
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,
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.
GC/MS was
performed on a Hewlett-Packard MS engine using both EI and CI
ionization modes.
Power-gated
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
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
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) .
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
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.
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
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
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
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
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
The presence of
phosphate in Ma1 and Ma2 and its location were investigated by NMR. Fig. 8represents the
From our previous study on the dManLAM structure, it
was assumed that the mannan core was composed by a backbone of (1
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.
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-
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
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
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
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
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.
We thank Dr. M. Gheorghiu for the M. bovis BCG cultures and G. Tuffal for technical assistance in the use of
pulsed amperometric detection.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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) .
-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) .
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.
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 D
O (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.
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).
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.
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.
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.
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).
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.
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.
-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 ().
-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.
-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) .
-Manp unit unsubstituted in positions
2 and 3.
-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.
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).
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.
-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.
-(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.
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.
-(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.
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.
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.