Variation in Mannose-capped Terminal Arabinan Motifs of
Lipoarabinomannans from Clinical Isolates of Mycobacterium
tuberculosis and Mycobacterium avium Complex*
Kay-Hooi
Khoo
,
Jyh-Bing
Tang
, and
Delphi
Chatterjee§¶
From the § Department of Microbiology, Colorado State
University, Fort Collins, Colorado 80523 and the
Institute of Biological Chemistry, Academia Sinica,
Taipei 115, Taiwan
Received for publication, May 10, 2000, and in revised form, October 27, 2000
 |
ABSTRACT |
The unique terminal arabinan motifs of
mycobacterial lipoarabinomannan (LAM), which are mannose-capped to
different extents, probably constitute the single most important
structural entity engaged in receptor binding and subsequent
immunopathogenesis. We have developed a concerted approach of
endoarabinanase digestion coupled with chromatography and mass
spectrometry analysis to rapidly identify and quantitatively map the
complement of such terminal units among the clinical isolates of
different virulence and drug resistance profiles. In comparison with
LAM from laboratory strains of Mycobacterium tuberculosis,
an ethambutol (Emb) resistant clinical isolate was shown to have a
significantly higher proportion of nonmannose capped arabinan termini.
More drastically, the mannose capping was completely inhibited when an
Emb-susceptible strain was grown in the presence of subminimal
inhibitory concentration of Emb. Both cases resulted in an increase of
arabinose to mannose ratio in the overall glycosyl composition of LAM.
Emb, therefore, not only could affect the complete elaboration of the
arabinan as found previously for LAM from Mycobacterium
smegmatis resistant mutant but also could inhibit the extent of
mannose capping and hence its associated biological functions in
M. tuberculosis. Unexpectedly, an intrinsically
Emb-resistant Mycobacterium avium isolate of smooth
transparent colony morphology was found to have most of its arabinan
termini capped with a single mannose residue instead of the more common
dimannoside as established for LAM from M. tuberculosis.
This is the first report on the LAM structure from M. avium
complex, an increasingly important opportunistic infectious agent
afflicting AIDS patients.
 |
INTRODUCTION |
One of the most prominent macromolecular entities of all
mycobacterial cell walls is the arabinan, a common constituent of both
the arabinogalactan (AG)1 and
lipoarabinomannan (LAM) (1). In the chemical setting of mycolylarabinogalactan-peptidoglycan complex, AG forms an integral part
of the cell wall proper, whereas the wall associated LAM has been
implicated as a key molecule involved in the virulence and
immunopathogenesis of tuberculosis (2). The arabinan of LAM is attached
to a mannan core that extends from a phosphatidylinositol mannoside
anchor at the reducing end. Of major biological significance is the
degree and chemical nature of capping functions and other substituents
on the arabinan. It was demonstrated that the arabinan chains of LAMs
from Mycobacterium tuberculosis, as well as the attenuated
Mycobacterium bovis BCG vaccine strain and
Mycobacterium leprae, the etiological agent of leprosy, are
mannose-capped to varying degrees (40-70%). In contrast, the majority
of the arabinan termini from the rapidly growing, noninfective
Mycobacterium smegmatis are uncapped, whereas a minor
portion terminates with inositol phosphate (3-6). The mannose-capped
LAM isolated from M. tuberculosis is generally less
effective at stimulating macrophages than the non-mannose-capped LAM
from fast growing strains (2), which may owe its potency in inducing
cytokine production entirely to its inositol phosphate capping (5, 6).
The presence of mannose caps on LAM has also been implicated to be
essential in mediating the binding to human macrophage mannose
receptors (7-9) and lung surfactant proteins (10, 11), thereby
affecting the adherence of whole bacterium to macrophage host and its
subsequent route of entry.
The arabinan represents a valid target for antimycobacterial drug
because disruption of its biosynthesis would lead to dual dismantling
of both the mycolyl-AG-peptidoglycan cell wall complex and LAM.
Ethambutol (Emb), an effective antimycobacterial drug known to inhibit
the biogenesis of the cell wall components, is now understood to act by
inhibiting the arabinan biosynthesis (12-14), but its precise site or
mechanism of action is still unknown. Interestingly, an Emb-resistant
mutant derived from M. smegmatis apparently made normal cell
wall AG but truncated LAM of smaller size in an Emb
dose-dependent manner when grown in the presence of this
drug (15). It is not known whether this phenomenon of truncation
extends to mannose-capped LAMs from M. tuberculosis, which
has a very similar underlying arabinan structure. Specifically, it is
of interest to know whether Emb inhibition of arabinan synthesis would
result in concomitant reduction in the extent of mannose capping on
prematurely terminated arabinan chains, thereby compromising their
biological activities.
LAM is a notoriously difficult biomolecule for precise structure and
function definition because of its size, extreme heterogeneity, and the
chemical nature of the arabinan. Further, it is not known whether our
current understanding based on LAM samples from well established
laboratory strains, such as M. tuberculosis H37Ra and H37Rv
or M. bovis BCG and M. smegmatis, truly reflects
in vivo scenario of infection. At present, virtually nothing
is known about the branching pattern and capping function of LAMs from drug-resistant clinical isolates that are difficult to obtain in large
quantities. Given the particular biological relevance of the terminal
arabinan structures, a rapid and high sensitivity method of defining
their relative profiles is imperative to further understanding of the
consequence of drug action and resistance. Toward this end, our own
crude endoarabinanase preparation (16) provides the only presently
available tool to release intact the variably capped terminal
oligoarabinosyl motifs from LAM (5, 17) without resorting to chemical
cleavage methods that would otherwise degrade the labile
arabinofuranosyl bonds.
We demonstrated in this paper that endoarabinanase digestion of intact
LAM followed by direct high pH anion exchange chromatography (HPAEC)
and mass spectrometry (MS) analysis can be a quick and effective way to
assess the branching nature and capping functions of the arabinan
motifs. For more detailed characterization, the enzymatically released
oligomers can be fluorescently labeled at the reducing end and
fractionated on normal phase HPLC. The liquid chromatography based
analysis afforded a useful quantitative map of various digested
products, whereas MS analysis allowed firm identification based on
molecular mass, along with any novel substituents. These two approaches
are therefore highly complementary and were used here to gain an
insight into the arabinan framework of each LAM under investigation. We
provided the first pictures of the native construct of the arabinan
terminal motifs in LAMs from clinical isolates of M. tuberculosis and M. avium complex and highlighted their
substantial differences in the nature of mannose capping. We also
demonstrated, for the first time, that mannose capping on LAM from the
laboratory M. tuberculosis strain can be completely
abolished when grown in the presence of Emb at a concentration that
would be ineffective against the drug resistant clinical isolates.
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EXPERIMENTAL PROCEDURES |
Chemical Reagents--
All chemical reagents were of highest
grade from Aldrich/Fluka unless otherwise specified. Milli-Q® water
was used for all chemical reactions and also for isolation of
lipoglycans and column chromatography.
Growth Conditions of Mycobacteria Isolates--
The
Emb-resistant M. tuberculosis isolate CSU19 (from Korea) was
propagated on 7H11 plates. Isolated colonies were then grown in 5 ml of
glycerol-alanine salts broth for 2-3 weeks at 37 °C on a shaker.
Large scale cultures were initiated by using 100 µl of broth culture
to inoculate 5 ml of fresh glycerol-alanine salts followed by 1 ml of
culture into 400 ml of broth. M. tuberculosis, H37Ra, was
grown in glycerol-alanine salts broth in the presence of 0.5 µg of
Emb. M. avium, strain 2151, was grown in Middlebrook 7H11
agar plates with oleic acid/albumin/dextrose/catalase enrichment supplement to maintain stable colony morphotypes (18). The smooth transparent (SmT) type was used in this study.
Extraction of LAM--
Wet cells (~10 g) were disrupted
mechanically using either a French Pressure Cell or a Bead Beater as
required for over 90% breakage of cells. The initial breaking buffer
for disruption contained Triton X-114 (Pierce) in phosphate-buffered
saline in the presence of a protease inhibitor mixture (pepstatin,
phenylmethylsulfonyl fluoride, and leupeptin) and DNase, RNase. The
suspension after cell breakage was centrifuged at 27,000 × g for 1 h at 4 °C. The cell pellet was resuspended
in breaking buffer and rocked overnight at 4 °C. The cell pellet was
resuspended in breaking buffer and centrifuged as before. The combined
supernatant was placed at 37 °C to initiate biphase (19) and
centrifuged at 12,000 × g for 15 min at room
temperature. The aqueous layer and the detergent layer were back
extracted a second time, the detergent layer was separated, and the
combined detergent layer was precipitated with 9 volumes of acetone.
The acetone precipitate was collected, dried, and partitioned between
phenol and water. The aqueous layer that contained majority of cellular
LAM and lipomannan was dried and used for further purification (3).
Size Fractionation and SDS-PAGE--
Sephacryl S-200 (Amersham
Pharmacia Biotech) column was prepared by using methods as described
before (15). The gel was suspended in a buffer containing 0.2 M NaCl, 0.25% deoxycholate, 1 mM EDTA, 0.02%
sodium azide, and 10 mM Tris, pH 8.0 (3). SDS-PAGE was used
to monitor the elution profile of fractions containing LAM and
lipomannan, which were then pooled accordingly and dialyzed at 37 °C
without detergent followed by water for several days. LAM/lipomannan
thus recovered was reanalyzed by SDS-PAGE to check for purity prior to
detailed analysis. SDS-PAGE and silver-periodic acid Schiff staining
were performed essentially as described (4). Sample concentrations were
maintained at 1-2 µg in 10 µl of sample buffer.
Digestion with Endoarabinanase and Subsequent
Analyses--
Selective growth of a soil microorganism,
Cellulomonas gelida, from which the endoarabinanase was
purified, has been described (16). The crude enzyme preparation
(containing both endo and exo glycosidases) was fractionated on a
Q-Sepharose column, and the eluents were examined for their ability to
digest AG. The active fractions were then applied to a Cibacron Blue
3GA Affinity column (Sigma) and eluted with a step gradient of
0.2-1 M NaCl. The fractions were then desalted
using Centricon, and enzyme activity was remonitored. The suitable
fractions were pooled and dialyzed overnight against 100 mM
Tris/HCl. After a final concentration of the enzyme dialysate in 20%
polyethylene glycol, the concentrated solution is stored and maintained
at 4 °C in the Tris buffer. The activity of the enzyme reduced
significantly when the initial dialysis was carried out in water as
opposed to the Tris/HCl.
To ensure a directly comparable and consistent result, all LAM samples
(10 µg for initial mapping; 50 µg for enzyme digestion and
subsequent analysis by 2-AB tagging and HPLC/MS analysis) were treated
with same amount of the enzyme preparation for 4 h at 37 °C. An
aliquot from the digestion product mixtures that contained both mannan
core and the released oligoarabinosides were analyzed directly by
HPAEC. Analytical HPAEC was performed on a Dionex LC system fitted with
a Dionex Carbopac PA-1 column (15), and the oligoarabinosides were
detected with a pulse amperometric detector (PAD-II). The remaining
sample was frozen and dried down in SpeedVac (Savant) for subsequent MS
analysis. For larger preparation, the reaction mixtures were boiled
briefly to terminate the digestions and desalted directly to remove the
Tris salts and the mannan core, as described in the "2-Ab
Labeling and Normal Phase HPLC".
2-AB Labeling and Normal Phase HPLC--
Endoarabinanase
released oligomers were first separated from resistant LAM core and
desalted simultaneously with a HyperSep Hypercarb poros graphitized
carbon (PGC) column (ThermoQuest). Samples were dissolved in water and
loaded onto the cartridge. After washing with 3 ml of water, the
oligosaccharides were eluted with 3 ml of 25% acetonitrile, whereas
the mannan core was recovered in the subsequent 3-ml fraction of 50%
acetonitrile containing 0.1% trifluoroacetic acid. The purified
oligosaccharides were then incubated with 5 µl of the labeling
reagents (0.35 M 2-aminobenzamide and 1 M
sodium cyanoborohydride in 70:30 dimethyl sulfoxide:glacial acetic
acid) at 65 °C for 2 h, after which the reaction mixtures were
directly spotted onto a GlycoClean S cartridge (Glyko). After 15 min,
the disc was washed sequentially with 1 ml of acetonitrile, 5× 1 ml of
96% acetonitrile, and finally the 2-AB-labeled glycans were recovered
by eluting with 3× 0.5 ml of water. Normal phase HPLC was performed on
a Hewlett Packard 1100 series LC system fitted with a thermostat column
compartment and a PalPak type N column (Takara, 4.6 × 250 mm).
Solvents A and B are acetonitrile and 250 mM aqueous formic
acid, respectively. 2-AB-labeled glycans were size fractionated with a
gradient of t = 0, 20% B, t = 40 min,
40% B, t = 65 min, 85% B, at a flow rate of 0.5 ml/min, and detected by fluorescence with excitation and emission
wavelengths set at 310 and 410 nm, respectively.
Chemical Derivatization and MS Analysis--
Samples were
permethylated using the NaOH/dimethyl sulfoxide slurry method as
described by Dell et al. (20). FAB-MS and ESI-MS analyses
were performed on an Autospec orthogonal acceleration-time of flight
mass spectrometer (Micromass, Manchester, UK), and fitted with a
magnet bypass flight tube and interchangeable FAB and ESI source
assemblies. For FAB-MS experiments, the fitted cesium ion gun was
operated at 26 kV, and the source accelerating voltage was operated at
8 kV. The permethyl derivatives were redissolved in CH3OH
for loading onto the probe tip coated with
m-nitrobenzylalcohol as matrix. Collision-induced
dissociation FAB-MS/MS was performed by introducing argon gas to the
collision cell to a reading of ~1.2 × 10
6
millibars on the time of flight ion gauge, at a lab frame collision energy of 800 eV and a push-out frequency of 56 kHz for orthogonal sampling. A 1-s integration time per spectrum was chosen for the time
of flight analyzer with a 0.1-s interscan delay. Individual spectra
were summed for data processing. For ESI-MS, the accelerating voltage
was maintained at 4 kV. The permethyl derivatives were dissolved in
CH3OH, and 10-µl aliquots were injected through a Rheodyne loop into the mobile phase (methanol/water/acetic acid, 50:50:1, v/v/v), delivered at a flow rate of 5 µl/min into the ESI
source by a syringe pump. Underivatized native samples analyzed by
ESI-MS were first subjected to clean up using the PGC cartridge described to remove salts and mannan core.
 |
RESULTS |
Our previous work has shown that the
1-5 arabinan chains of
LAM can be digested by endoarabinanase from C. gelida into
di-arabinosides, Araf
1
5Araf
(Ara2). However, the characteristic chain termination with
a Araf
1
2Araf renders the adjacent
Araf
1
5Araf unit resistant to
endoarabinanase digestion, leading instead to the recovery of variably
capped nonreducing terminal tetra- (Ara4) and
hexa-arabinosyl (Ara6) units (Fig.
1). Thus, for an extensively
mannose-capped LAM such as that from M. tuberculosis Erdman,
the major products have been isolated by size fractionation on a
Bio-Gel P-6 column and structurally characterized as Ara2,
Man2Ara4, and Man4Ara6 because the most abundant cap on each arabinan terminus is
Man
1
2Man (17). On the other hand, endoarabinanase digestion of
non mannose-capped LAM gave Ara2, Ara4, and
Ara6 as major products (5).

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Fig. 1.
The nonreducing terminal oligoarabinosyl
structural motifs of LAM. The formation of the characteristic
mannose-capped linear Ara4 and branched Ara6
motifs (boxed) by endoarabinanase cleavages are indicated by
vertical down arrows at site I. 2-Ara chain termination
with and without additional 3-branching will render site II
resistant to cleavage. The remaining of the arabinan chains are mostly
digested into Ara2. Heterogeneity in digestion products
mainly reflects the heterogeneity in the size of the mannose cap on
each terminus.
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HPAEC Mapping of Endoarabinanase Digestion Products--
LAM from
M. tuberculosis H37Rv (RvLAM) has previously been shown to
be very similar in structure to that from Erdman, although the relevant
ManxAran terminal units have not been isolated intact.
To rapidly map these nonreducing terminal motifs, the endoarabinanase
digested RvLAM was analyzed directly by HPAEC, and the profile obtained
(Fig. 2A) was compared against that from the same sample further treated with
-mannosidase to remove the caps (Fig. 2B). The latter was found to give a
simpler profile with three major peaks, which eluted at retention times corresponding to Ara2, Ara4, and
Ara6 standards (peaks 1-3 in Fig.
2B). Thus, the major endoarabinanase digestion products of RvLAM as observed in Fig. 2A could be assigned as
mannose-capped Ara4 and Ara6. This was further
corroborated when Man2Ara4 and Man4Ara6 isolated from a Bio-Gel P-6 column and
identified by MS analysis were found to elute at positions
corresponding to peaks 4 and 5, respectively (data not shown). The
major mannose-capped Ara4/Ara6 motifs, namely
Man2Ara4 and Man4Ara6,
could therefore be adequately resolved from the uncapped
Ara4 and Ara6, respectively. Interestingly,
mannose substitution at 5-OH of the
-Ara results in earlier
retention time of the oligoarabinosyl motifs, indicating that
separation by HPAEC is both structure- and size-dependent. Direct analysis by HPAEC of the endoarabinanase digested LAM will thus
give a rapid indication of whether the LAM under investigation does
terminate with the Ara4/Ara6 motifs and whether
it is characteristically capped with a dimannoside.

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Fig. 2.
HPAEC profiles of the endoarabinanase
digestion products of RvLAM (A) and after
-mannosidase digestion (B).
Peak 1, Ara2; peak 2,
Ara4; peak 3, Ara6; peak
4, Man2Ara4; peak 5,
Man4Ara6. The huge peak at around 6 min in
B is mannose residue released by -mannosidase.
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MS Profiling of Endoarabinanase Digestion Products--
To provide
a more definitive identification of the oligoarabinosyl motifs, the
digestion product mixture of RvLAM was dried down directly,
permethylated, chloroform extracted to remove most of the salt buffers
used, and analyzed by MS. Rather unexpectedly, the
m/z values of the major molecular ions did not
correspond to the calculated molecular masses of the expected molecular
species. Instead, they could be assigned as incorporating a Tris
moiety at the reducing end. Thus, the expected [M+Na]+
molecular ions for Man2Ara4 and
Man4Ara6 are only observed as minor signals at
m/z 1117 and 1845, respectively, whereas the corresponding ones assigned as incorporating a Tris adduct afforded strong M+ signals at m/z 1254 and
1982 (Fig. 3A). Other
Tris-incorporating molecular ion signals observed include
Ara2, Ara4, Ara6,
Man3Ara4, Man2Ara6, and
Man5Ara6 at m/z 526, 846, 1166, 1458, 1574, and 2186, respectively. The assigned molecular
composition clearly demonstrated that most of the heterogeneity
associated with the digestion products could be attributed to the
mannose capping, which ranges from Man1 to Man3
or no capping on each terminus. MS/MS analysis of the
Man2Ara4 peak at m/z 1254 yielded a series of fragment ions at m/z 380, 540, 700, 860, and 1064, corresponding to reducing terminal ring
cleavage ions Ara1 + Tris, Ara2 + Tris, Ara3 + Tris, Ara4 + Tris, and
Man1Ara4 + Tris, respectively (Fig. 3B). Together with additional fragment ions, the MS/MS data
supported a linear Man2Ara4 structure as drawn
(Fig. 3B) and also indicated a covalent incorporation of
Tris at the reducing terminus.

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Fig. 3.
MS analysis of the endoarabinanase-digested
RvLAM. A, the reaction mixtures were dried down and
permethylated directly for FAB-MS analysis. B, FAB-MS/MS
analysis of the parent ion at m/z 1254 from
A with a schematic drawing showing the probable cleavage
pathways. The insets show the ESI-mass spectra for the
native samples, focusing on the two major components i.e.
Man2Ara4 and Man4Ara6,
to show the effects of sample processing on Tris incorporation. Samples
which were dried down first (inset C) have a high propensity
to incorporate a Tris molecule coming from the concentrated
enzyme buffer. Signals at m/z 996, 1584, and 1746 correspond to [M+Na]+ of Tris-incorporating
Man2Ara4, Man4Ara6, and
Man5Ara6, respectively. The adducts could be
avoided if the reaction mixtures were first passed through a PGC column
to remove the salts prior to drying down and analyzed by MS
(inset D). The [[M+Na]+ signals were shifted
to 103 units lower as compared with (inset C). The
schematic drawing showing Tris incorporation at the reducing end only
serves to illustrate how it affects the m/z
values observed. The exact chemical structures have not been proven,
and Tris may also be transglycosylated through one of the OH groups
instead of the amine.
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Further investigation demonstrated that Tris incorporation could be
largely avoided if the enzyme digested mixture was first passed through
a PGC column for salt removal instead of drying down directly, which
probably concentrated the Tris buffer while the enzyme was still active
(Fig. 3, insets C and D). Thus, for a specific
purpose when it is imperative to maintain the free reducing end or for
a large scale preparation, the digestion products could be desalted
directly using a PGC or gel filtration column, which also render the
sample clean enough for direct ESI-MS analysis (Fig. 3D).
However, when sample amount is limited, permethylation of the dried
down digestion products is preferable because it avoids the additional
step of work up and improves sensitivity of detection by MS, probably
because of creation of a fixed charge on the quaternary ammonium during
permethylation. Thus, although it is yet unclear how the released
oligoarabinosyl motifs could be transglycosylated with Tris present in
the enzyme buffer, its stable incorporation clearly did not interfere
with MS analysis. In fact, the dried down sample was equally amenable
to further clean up with PGC column for ESI-MS analysis (Fig.
3C). The m/z values of the observed
[M+Na]+ molecular ion signals for the Tris incorporating
native samples further validated our assignment of the molecular
compositions of the various oligoarabinosyl motifs.
Emb and Mannose Capping in M. tuberculosis LAM--
As shown
previously, LAM from the avirulent H37Ra (RaLAM) was very similar to
that from the virulent Erdman or H37Rv strain in that it was also
mannose-capped. The HPAEC and MS profiles of endoarabinanase digested
RaLAM (Fig. 4A) were indeed
similar to those of RvLAM, with Man2Ara4
(m/z 1254) and Man4Ara6
(m/z 1982) being the dominant products. Other
molecular ions afforded by ESI-MS analysis of the permethyl derivatives
attested to the variation in mannose capping, corresponding to
Ara4 (m/z 846) with shorter
(Man1Ara4, m/z 1050) and
longer (Man3Ara4, m/z
1458) mannose caps and Ara6 with a total of 0, 2, 3, 5, and
6 Man residues at m/z 1166, 1574, 1778, 2186, and
2390 respectively. In addition, Ara5
(m/z 1006) and Man2Ara5
(m/z 1414) were also detected.

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Fig. 4.
HPAEC and MS analysis of the
endoarabinanase-digested RaLAM. A, ESI-mass spectrum of
the permethyl derivatives of digested RaLAM from H37Ra grown under
normal conditions. The inset shows the direct HPAEC profile
of the digestion products. B, ESI-mass spectrum of the
permethyl derivatives of digested RaLAMemb from H37Ra grown
in the presence of 0.5 µg of Emb. The inset shows the direct HPAEC
profile of the digestion products (upper panel) as compared
with that of normal RaLAM further treated with -mannosidase
(lower panel). The HPAEC peaks 1-5 are as assigned for
RvLAM (Fig. 2). The identity of the late eluting peak in the HPAEC
profile of RaLAMemb is unknown at present but was
unaffected by further -mannosidase treatment.
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Interestingly, the RaLAM derived from H37Ra propagated and maintained
in an Emb concentration of 0.5 µg/ml (minimal inhibitory concentration = 1 µg/ml) (RaLAMemb) afforded a HPAEC
profile that resembled that of
-mannosidase-treated samples, namely,
instead of the ManxAran peaks, RaLAMemb
yielded mainly the noncapped Ara4 and Ara6
(Fig. 4B, inset). In comparison with
-mannosidase-treated normal RaLAM, the amount of Ara4
and Ara6 terminal motifs were clearly reduced relative to
Ara2, indicating that a larger proportion of the arabinan
of RaLAMemb terminated prematurely, concomitant with loss
of mannose capping. In support of the HPAEC analysis, glycosyl
composition analysis showed that the Ara:Man ratio for
RaLAMemb was 2.03, whereas that of a "normal" RaLAM was
0.95. ESI-MS analysis of the permethyl derivatives of the digestion
products further confirmed that the major oligoarabinosyl motifs
detected were devoid of Man capping (Fig. 4B). In addition to Ara2, Ara4, and Ara6,
significant amounts of Ara7 (m/z
1326), Ara8 (m/z 1486), and larger
oligoarabinosides (Ara9 up to at least Ara14)
were also detected. Together, the data clearly showed that application
of 0.5 µg/ml of Emb to the M. tuberculosis, H37Ra culture
has drastically led to complete inhibition of mannose capping,
concomitant with significant alteration in the underlying arabinan framework.
Mannose Capping in Drug-resistant Clinical Isolate of M. tuberculosis--
The LAM isolated from Emb-resistant CSU19 clinical
isolate (TbLAM) grown without any Emb showed endoarabinanase
digestion/HPAEC and MS profiles (Fig. 5)
distinct from that of RvLAM. TbLAM was deduced to be more heterogeneous
in the spread of size, ranging from full size to severely truncated
even in the absence of Emb (results not shown). Its average Ara:Man
molar ratio of 1.44 is somewhat intermediate between a normal
mannose-capped LAM (~1.0) and the noncapped RaLAMemb
(2.03). The HPAEC profile of its endoarabinanase digestion products
indicated that peaks corresponding to Man2Ara4 (peak 4) and Ara4 (peak 2) were almost of equal abundance,
whereas the Man4Ara6 peak (peak 5) was reduced
in relative abundance (Fig. 5, inset). Interestingly, after
-mannosidase digestion, the relative ratio of the resultant
Ara2, Ara4, and Ara6 peaks was
comparable with that of RvLAM and RaLAM, indicating that the difference
was mainly due to mannose capping. FAB-MS analysis of the TbLAM
digestion products corroborated this observation (Fig. 5).
Qualitatively, all peaks observed for RvLAM were also present in TbLAM
but with significant changes in relative intensities. Most obvious was the enhanced abundance of molecular ions corresponding to
Ara4 (m/z 846) relative to
Man2Ara4 (m/z 1254),
whereas Man4Ara6 (m/z 1982) was significantly reduced, and Man5Ara6
was hardly detected.

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Fig. 5.
HPAEC and MS analysis of the
endoarabinanase digested TbLAM. The reaction mixtures were dried
down and permethylated directly for FAB-MS analysis. The
inset shows the direct HPAEC profile of the digestion
products (upper panel) as compared against that of the same
sample further treated with -mannosidase (lower panel).
The HPAEC peaks 1-5 are as assigned for RvLAM (Fig. 2).
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To further confirm the observed changes in the relative amounts of the
ManxAran oligomers, the endoarabinanase digestion
products were subjected to fluorescent tagging with 2-AB after removal
of the resistant lipomannan core and other salt contaminant including
Tris, which would affect the reducing end tagging. The 2-AB-tagged
products were then size fractionated by normal phase HPLC on an amine
column. Each major peak detected was collected and analyzed by MS to
confirm its identity by molecular mass (Table
I). In comparison with similarly analyzed
RaLAM (Fig. 6A), the HPLC
profile of TbLAM (Fig. 6C) clearly showed that the peak
corresponding to Ara4 (peak 2) was much enhanced in
abundance relative to Man2Ara4 (peak 6) and
that both Man4Ara6 (peak 8) and
Man5Ara6 (peak 9) were reduced in abundance. MS
analysis also showed that peak 7 for RaLAM was
Man3Ara4, whereas the corresponding peak 7' for
TbLAM was found to contain mostly Man2Ara6. For
RaLAMemb (Fig. 6B), in agreement with the HPAEC
and MS data (Fig. 4), all mannose-capped oligoarabinoside peaks (peaks
6-9) were absent. Instead, Ara7 (peak 4) and
Ara8 (peak 5) were identified as additional peaks not found
in RaLAM or TbLAM.
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Table I
Identification and quantitation of 2-AB-tagged ManxAran
oligomers produced by endoarabinanase digested LAM from M. tuberculosis and fractionated by normal phase HPLC (Fig. 6)
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Fig. 6.
Normal phase HPLC profiles of the 2-AB
derivatives of the endoarabinanase digested products of RaLAM
(A), RaLAMemb (B), and
TbLAM (C). Peaks were detected by fluorescence,
and those numbered 1-9 were identified by MS analysis (Table I).
Because of a prior clean up of the endoarabinanase digestion products
by passing through a PGC cartridge, a significant amount of
Ara2 was lost from the retained fractions used for 2-AB
tagging and HPLC. Peak 1 was additionally contributed by other
fluorescent contaminants and therefore could not be used as a
quantitative representation of Ara2.
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Because the detector response of fluorescently labeled oligomers was
based on the fluorophore incorporated at the reducing end of each
molecule, it is relatively independent of the overall size or structure
of the oligosaccharides. The HPLC profile therefore provided a more
accurate representation of the relative abundance of each
oligoarabinosyl motifs in comparison with the HPAEC profile, where the
response factor is more structure-dependent. Integration of
the areas for each major peak detected therefore provided a way to
estimate their relative abundance. It was found that the molar ratio of
Man2Ara4 to Ara4 for TbLAM was
about 1:1 (50% capping for the linear Ara4 termini),
whereas in RaLAM, the total of Man2Ara4 and
Man3Ara4 relative to noncapped Ara4
was 17.6:1, or approximating 95% capping. For the branched
Ara6 termini, the total of Man4Ara6 and Man5Ara6 relative to uncapped
Ara6 (peak 3) in RaLAM was 2.35:1 or about 70% capping. In
contrast, the total of mannose-capped Ara6
(Man2Ara6, Man4Ara6,
and Man5Ara6) relative to noncapped Ara6 in TbLAM was 1.2:1 or about 55% capping. Based on
these estimations, the total Ara4:Ara6 ratio
(capped and uncapped) in RaLAM was ~0.74, whereas in TbLAM the ratio
was 1.4. The comparative figure for RaLAMemb was 0.45. These calculated figures did not take into consideration other minor
peaks or cases where mannose-capped Ara4 and
Ara6 coeluted together within a single fluorescently detected peak, for example peak 7' (Fig. 6C). Nevertheless,
the major conclusion derived from three independent approaches was fairly consistent. It can be seen from the HPAEC profile of
-mannosidase-treated samples that for RaLAM, the Ara6
peak was marginally more abundant than the Ara4 peak,
whereas the reverse is true for TbLAM. For "normal" RaLAM, the
Ara4 termini was highly capped (95%), whereas the degree
of mannose capping in Ara6 termini was only 70%, therefore yielding a significant amount of noncapped Ara6 in the
digestion products. For the Emb-resistant clinical isolate, both
termini were only 50-55% mannose-capped. A slightly lower degree of
branching was also observed, which gave rise to more linear
Ara4 relative to Ara6.
Single Mannose Capping in Clinical Isolate of M. avium--
The
HPAEC profile of digested LAM from a clinical isolate of M. avium (AvLAM) (Fig. 7B)
indicated that it was rather different from the M. tuberculosis LAM described above. However, the same characteristic
Ara2, Ara4, and Ara6 peaks were
produced after
-mannosidase digestion (Fig. 7C),
suggesting that the underlying arabinan motifs remain similar and that
structural difference may again reside on the variation in mannose
capping. Further information was derived from MS analysis (Fig.
7A) in conjunction with HPLC fractionation of the
2-AB-tagged digestion products (Fig. 7D). The single most
important difference as revealed by ESI-MS analysis of the total
digestion products was the dominance of
Man1Ara4 (m/z 1050) with
very little Man2Ara4 (m/z
1254) detected. Of Ara6-containing species, the most
prominent were Man1Ara6
(m/z 1370) and Man2Ara6
(m/z 1574), although
Man3Ara6 (m/z 1778),
Man4Ara6 (m/z 1982), and
Man5Ara6 (m/z 2186) could
also be detected. This was well reflected by the normal phase HPLC
profile of the 2-AB-tagged sample (Fig. 7D). The most
abundant peak 3' was clearly shown by FAB-MS and MS/MS analysis to be
2-AB-tagged Man1Ara4 with a fragmentation
pattern consistent with a linear Ara4 capped at the
nonreducing end by a single Man (Fig.
8C). Importantly, the fragment
ions at m/z 219 (Hex+) and 187 (
Hex+) indicated that the nonreducing terminal was a Man
and not Ara, which would otherwise give prominent fragment ions at
m/z 175 (Pent+) and 143 (
Pent+) as afforded by Ara4 (Fig.
8A) and Ara6 (Fig. 8B). These
nonreducing terminal oxonium ions were characteristically very
abundant, even in the daughter ion spectrum of other weaker parent
ions, which did not afford any other significant fragment ions apart
from m/z 355 (HO)Ara1-AB. Thus,
additional nonreducing terminal ion at m/z 391 corresponding to
Hex-Hex+ was afforded by
Man2Ara4 and indicated that it was a linear
hexamer with a dimannoside at the nonreducing end (Fig. 8D).
Interestingly, only fragment ions at m/z 187 and
219 were detected for Man2Ara6 (Fig.
8E) and not ions that would correspond to terminal
Ara+ (m/z 175/143) or
Man2+ (m/z 391). The
latter ion was, however, present in the MS/MS spectrum of
Man4Ara6 (Fig. 8F). It may thus be
concluded that the arabinan in AvLAM terminate in the same way as
either the linear Ara4 or branched Ara6 motifs.
The amount of Ara2 relative to the terminal
Ara4 and Ara6 was, however, not as high as in LAM from M. tuberculosis, as shown by the HPAEC profile of
-mannosidase-treated sample (Fig. 6C). Very little of
these termini were exposed. Instead, our data suggested that most were
capped with a single mannose residue, yielding
Man1Ara4 and Man2Ara6
as the most abundant endoarabinanase digestion products, although
Man2 cap was also found.

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Fig. 7.
Analysis of the endoarabinanase digested
AvLAM. A, the reaction mixtures were dried down and
permethylated directly for ESI-MS analysis. B, the direct
HPAEC profile of the digestion products as compared against that of the
same sample further treated with -mannosidase (C). The
HPAEC peaks 1-5 are as assigned for RvLAM (Fig. 2). D, the
normal phase HPLC profile of the 2-AB derivatives of the digested
products after a further PGC column clean up. The numbering of the
peaks is as in Fig. 6. A prime is used to distinguish peaks of same
retention time containing different or additional components:
Peak 3', mainly Man1Ara4 with small
amount of Ara6; peak 6',
Man1Ara6 and Man2Ara4;
peak 7', contains mainly
Man2Ara6.
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Fig. 8.
FAB-MS/MS analysis of the permethyl
derivatives of 2-AB-tagged Ara4 (A),
Ara6 (B), Man1Ara4
(C), Man2Ara4
(D), Man2Ara6
(E), and Man4Ara6
(F) from AvLAM. For the latter two, only the low
mass regions containing the diagnostic fragment ions were shown.
Because of very weak parent ions, other fragment ions were either too
weak or absent. Sections of the mass spectra are magnified to show the
detected peaks more clearly. Signals at m/z 143, 175, 187, 219, and 391 correspond to nonreducing terminal oxonium ions
as described in the text. Other ions are reducing terminal -cleavage
or ring cleavage ions. Man0,1,2Ara4 afforded a
(OH)1Ara-Ara-AB ion at m/z 515, which
could not be produced from ManxAra6 because the
second Ara from the 2-AB-tagged end is the branch point in the
latter.
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DISCUSSION |
In summary, we demonstrated that direct endoarabinanase digestion
followed by HPAEC analysis could rapidly give a first indication as to
the presence or absence of the expected ManxAran motifs. Using the well characterized RvLAM as the standard reference, a
"normal" mannose-capped LAM is expected to yield predominantly Ara2, Man2Ara4, and
Man4Ara6 as the endoarabinanase digestion products. Subsequent treatment with
-mannosidase would confirm that
both the linear Ara4 and branched Ara6 are
indeed synthesized and that their relative ratios could be compared
with that of RvLAM. A departure from this norm could be rapidly
fingerprinted, as demonstrated with analysis of other LAMs reported
here. However, because of complexity and heterogeneity of the products
that would result from endoarabinanase digestion, a further
confirmatory analysis such as that using MS is required, particularly
to define the unknown peaks produced when analyzing a whole
spectrum of LAMs from different sources.
For MS analysis, we found that permethylation followed by a simple
organic phase partition was sufficient to yield good quality spectrum.
Of the ionization methods tried, FAB-MS and ESI-MS both yielded
comparable molecular ion profiles with the latter affording better
signal to noise ratio especially for larger components. However, the
relative abundance would depend on the cone voltage employed, which
could be tuned to favor the high mass or low mass components. ESI-MS
could also be used to analyze native samples, but in general, all
samples for ESI-MS should be desalted prior to injection. The presence
of Tris in the incubation buffer could result in its covalent
incorporation into the digestion product if the mixtures were
concentrated and dried down directly prior to further processing. It is
as yet unclear what is the mechanism or the degree of incorporation
that appeared to vary from batch to batch. However, as demonstrated
here, this did not interfere but actually facilitated the MS analysis.
By creating a fixed charge through permethylation, the various
Tris-containing species ionized as M+ instead of the usual
M+Na+ molecular ions normally afforded by ESI-MS and FAB-MS
when the matrix was not doped with acid. Collision-induced dissociation MS/MS analysis of the permethyl derivatives of Tris-containing species
did not result in dissociation of the Tris moiety but yielded a series
of reducing terminal fragment ions retaining the Tris and hence the
charge. Additional ESI-MS analyses of native samples further showed
that Tris incorporation was not induced by permethylation. It should be
noted that probably only a fraction of each of the digestion products
actually had Tris covalently incorporated at the reducing end but
nevertheless ionized preferably over the non-Tris containing ones and
hence appeared to be the dominant molecular ions in both FAB- and
ESI-mass spectra. We further found that the Tris-containing species
could be reduced in the same way the non-Tris containing species got
reductively aminated with 2-aminobenzamide. After reducing end tagging,
both 2-AB-tagged and Tris-tagged species could be detected directly or
after permethylation by MS analysis. To avoid formation of Tris-tagged
species, it is necessary to first desalt the digestion mixtures prior
to drying down, which would minimize the Tris incorporation and
leave the reducing end free for maximum and quantitative tagging with 2-AB.
Although the quantitative yield of 2-AB tagging using the well
established protocols for glycans from glycoproteins (21) has not been
rigorously proven for the oligoarabinosides, the HPLC profile of the
resultant products based on fluorescence detection agreed well with
that by HPAEC PAD response. The 2-AB-tagged oligomers can be
fractionated by normal phase HPLC and conveniently collected for
further analysis without any desalting step because only volatile buffer system was used. In comparison with HPAEC using the Dionex LC
system, we achieved a better separation of the noncapped Aran peaks from those that were mannose-capped. In HPAEC, Ara4
and Man2Ara4 eluted close to one another,
whereas Man1Ara4 eluted in between. In the HPLC
of 2-AB-tagged sample, Man1Ara4 was not well
resolved from Ara6. LC mapping by either mode is therefore insufficient but, when complemented by MS screening, proves to be
highly effective for quantitative comparison of the various structural
motifs. Although the 2-AB derivatives could be analyzed native by MS,
we prefer the permethyl derivatives for better sensitivity and to
facilitate interpretation of MS-MS data.
Using these approaches, we demonstrated that LAMs from Emb-resistant
M. tuberculosis and M. avium clinical isolates
showed a significant reduction in Man2-capped
Ara4 and Man4-capped Ara6 motifs.
In the case of TbLAM, the preferred cap is still Man2, but
more arabinan termini, especially Ara4, were exposed. In
M. avium LAM, the preferred cap appeared to be
Man1. Consequently, Man1Ara4 and
Man2Ara6 appeared as the major digestion
products. However, up to Man3Ara4 and
Man4Ara6 could be detected. In contrast, no
mannose capping could be detected in LAM obtained from the H37Ra strain
grown in the presence of Emb. All LAMs do maintain the basic framework
of the arabinan structural motifs, as revealed by
-mannosidase
removal of the mannose caps.
The inhibitory effect of Emb on the biosynthesis of arabinan have been
demonstrated at several levels (12, 14, 22), whereas the phenomenon of
truncated LAM was reported for the fast growing, Emb-resistant M. smegmatis grown in the presence of Emb (15). Our studies on LAMs
from M. tuberculosis clinical isolates as reported here
represent the first of a series of work initiated to ascertain the
relationship, if any, between their arabinan structure and Emb
resistance. Our results clearly showed that variation in mannose
capping exists among different mycobacteria isolates that can be
further affected by the application of Emb during growth. Because
mannose capping would only be effected on a properly
-Ara terminated
chain, it can be envisaged that inhibited elongation and proper
termination of arabinan chains would also inhibit subsequent mannose
capping. It remains to be established whether such reduction in mannose
capping is wide spread among M. tuberculosis isolates and
how it may affect the dissemination and survival of this intracellular
pathogen of macrophage, not least in the context of mediating binding
to macrophage mannose receptor or lung surfactant proteins (9, 10, 23).
Even more intriguing is how it may translate into Emb resistance. The reduction in mannose capping in the presence of Emb argues that Emb
does perturb the machinery for a complete assembly of the arabinan in
LAM. It follows that an Emb-resistant strain must therefore be able to
somehow encounter this effect by altering one or more parts of this
machinery that could conceivably lead indirectly to alteration in
mannose capping.
The mechanism of drug resistance in M. avium complex is
further complicated by the additional presence of the strain-specific glycopeptidolipids (GPL) and different colony morphotypes when grown on
solid media, namely, smooth opaque (SmO), SmT, and rough. The
search for the basis of morphological variations in M. avium has been met with little enlightenment. It has been shown that the
rough strains are devoid of GPLs, whereas SmT and SmO forms express
these GPLs in similar quantities. In addition, SmT morphotype is
usually more virulent and shows more innate drug resistance than the
SmO type. Although the GPLs of M. avium have been studied extensively and have been implicated in drug resistance and
impermeability of the organism, structural or functional studies on LAM
have never been undertaken prior to this study. It is not known whether the predominance of single Man as the preferred cap in LAM from the
strain 2151 SmT investigated here is common among different M. avium strains of distinct colony morphotypes and drug resistance profiles. From the limited data obtained thus far, it would seem that
different mycobacteria species evolve different resistance mechanism in
neutralizing the inhibitory effect of Emb on the assembly of their
quintessential arabinan. Variation in mannose capping may be a
manifestation or consequence of such adaptation but with important
implications on their presumed biological roles.
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ACKNOWLEDGEMENTS |
We thank Israel Muro and Jerry Tews for
growth of the M. tuberculosis, H37Ra, and CSU 19 strains and
isolation and purification of LAM described in this manuscript. We also
thank Dr. Yi Xin for providing the endoarabinanase arabinan-degrading enzyme.
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FOOTNOTES |
*
This work was supported by Grants AI-37139 and TW 00943 from
the NIAID, National Institutes of Health (to D. C.) and in part by
Academia Sinica and National Science Council (Taipei, Taiwan) Grant NSC
89-2311-B-001-090 (to K. H. K.).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.:
970-491-7495; Fax: 970-491-1815; E-mail:
delphi@lamar.colostate.edu.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M004010200
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ABBREVIATIONS |
The abbreviations used are:
AG, arabinogalactan;
2-AB, 2-aminobenzamide;
Emb, ethambutol;
ESI, electrospray ionization;
FAB, fast atom bombardment;
GPL, glycopeptidolipid;
HPAEC, high pH
anion exchange chromatography;
LAM, lipoarabinomannan;
MS, mass
spectrometry;
PGC, poros graphitized carbon;
SmO, smooth opaque;
SmT, smooth transparent;
AvLAM, LAM from M. avium 2151;
RaLAM, LAM from M. tuberculosis H37Ra;
RvLAM, LAM from M. tuberculosis H37Rv;
RaLAMemb, LAM from M. tuberculosis H37Ra grown in the presence of Emb;
TbLAM, LAM from
M. tuberculosis CSU19;
HPLC, high pressure liquid
chromatography.
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.