From the
Previous studies have demonstrated that the nonreducing termini
of the lipoarabinomannan (LAM) from Mycobacterium tuberculosis are extensively capped with mannose residues, whereas those from a
fast growing Mycobacterium sp., once thought to be an
attenuated strain of M. tuberculosis, are not. The noncapped
LAM, termed AraLAM, is known to be more potent than the mannose-capped
LAM (ManLAM) in inducing functions associated with macrophage
activation. Using a combination of chemical and enzymatic approaches
coupled with fast atom bombardment-mass spectrometry analysis, we
demonstrated that LAMs from all M. tuberculosis strains
examined (Erdman, H37Ra, and H37Rv), as well as the attenuated
Mycobacterium bovis BCG strain, are mannose-capped with the
extent of capping varying between 40 and 70%. The nonreducing termini
of LAM from Mycobacterium leprae were also found to be capped
with mannoses but at a significantly lower level. A novel inositol
phosphate capping motif was identified on a minor portion of the
otherwise uncapped arabinan termini of LAMs from the fast growing
Mycobacterium sp. and Mycobacterium smegmatis ATCC
14468 and mc
Lipoarabinomannan (LAM)
Initial structural studies
demonstrated that the nonreducing termini of the arabinan of LAM from
the virulent M. tuberculosis Erdman strain, whether branched
or linear, were capped extensively (>70%) with Man residues (termed
ManLAM) (Chatterjee et al., 1992b) (Fig. S1). In
contrast, those from an ``avirulent'' fast growing strain,
which was believed at the time to be a rapidly growing M.
tuberculosis H37Ra variant, were uncapped (termed AraLAM)
(Chatterjee et al., 1991). It was then thought that these
terminal Man residues might be responsible for the differences in the
capacity of the two LAMs to induce functions associated with macrophage
activation and thus be related to virulence (Chatterjee et
al., 1992c; Roach et al., 1993, 1994). However, the
implication of the mannose caps as virulence factors was not supported
by subsequent observation that LAMs from both virulent M.
tuberculosis H37Rv and avirulent Mycobacterium bovis BCG
strains were mannose-capped (Prinzis et al., 1993; Venisse
et al., 1993). Furthermore, doubts were raised concerning the
identity of the highly passaged, fast growing strain from which AraLAM
was first isolated with the observation that it clearly was not an
authentic strain of M. tuberculosis H37Ra (Prinzis et
al., 1993).
Diazomethane was
generated from 1-methyl-3-nitro-1-nitrosoguanidine (Aldrich) in a 13
As described previously (Tsai et al., 1986),
each glycan fragment produced by acetolysis can exist in several
different forms depending on the extent of ring opening (Rosenfeld and
Ballou, 1974). Thus, in the first 30-40 min, deuteroacetolyzed
Man
LAMs from
different strains were subjected to mild trifluoroacetic acid
hydrolysis under conditions which were expected to specifically
hydrolyze furanosyl or glycosyl phosphate bonds (McConville and
Blackwell, 1991). After perdeuteroacetylation, the partial hydrolysates
were analyzed by FAB-MS in both positive and negative ion modes. For
all mannose-capped LAMs, positive ions corresponding to
Man
In the negative ion mode, the partial
hydrolysates of AraLAM gave prominent negative ion signals which were
absent in the negative ion spectra of all other LAMs. After further
separation of the hydrolysis fragments from the nonhydrolyzed
phosphatidylinositol-mannan core, the negatively charged components
from AraLAM afforded the negative FAB-mass spectrum shown in
Fig. 3
. The major molecular ion at m/z 751 corresponded
to (M - H)
CAD on
Ara
The Ino-P-Ara
In the negative FAB-mass spectrum of
the perdeuteroacetyl derivatives, (M - H)
As a complement to our earlier work on LAM involving detailed
chemical analysis of small fragments derived from partial hydrolysis of
per-O-alkylated LAMs (Chatterjee et al., 1991, 1992b;
Prinzis et al., 1993), alternative ways of characterizing the
nonreducing termini of LAM isolated from various strains of
Mycobacterium have now been developed based on FAB-MS analysis
of endoarabinase digestion and acetolysis products.
Peracetylation
of a variety of glycans under basic conditions retains most of the
common non-glycosyl substituents, notably acyl, phosphate, and sulfate
(Dell et al., 1994). Direct FAB-MS analysis of derivatized
sample prior to further purification allows rapid determination of the
exact molecular weight and hence the composition of each component
present in a mixture without prior knowledge of its chemical nature.
Despite being more complicated in spectra interpretation due to the
+dAc
In this
study, we reported the identification of a unique inositol phosphate
capping motif apparently confined to LAMs from rapidly growing
mycobacteria which are devoid of the mannose cap commonly present on
LAMs from M. tuberculosis. Our results suggested that the
inositol phosphate cap is probably restricted to a portion of the
linear arabinan termini while the unsubstituted branched arabinan
termini, which are probably the immunodominant epitopes, constitute the
bulk of AraLAM. It should be emphasized that inositol phosphate caps
only a minor portion of the arabinan termini, the majority of which are
exposed as evident from the terminal Ara:2-Ara ratio of 7:9 in our
methylation analysis of AraLAM. In retrospect, this novel entity may
actually correspond to the ``base-labile inositol phosphate''
described previously by Hunter et al.(1990). The low abundance
of this motif also presents considerable challenge to its complete
characterization. Preliminary linkage and CAD MS/MS data suggested that
the inositol phosphate is linked to position 5 of the terminal
It is now clear that AraLAM from the
dubious, rapidly growing Mycobacterium sp. is distinctively
different from LAM from a genuine M. tuberculosis H37Ra strain
which is of the ManLAM type. In addition, LAM from well known strains
of the rapidly growing M. smegmatis was shown to be similar to
AraLAM and that the same Ino-P-Ara
The earlier studies on the greater potency of AraLAM over ManLAM in
inducing a macrophage TNF-
The
structural studies reported in this work provide a foundation for
future research directed toward probing the function of inositol
phosphate capping of the terminal Araf residues. For instance,
it will be interesting to further determine whether this particular
modification of LAM contributes toward induction of different cytokines
other than TNF, to ascertain if it is responsible for inducing early
gene responses, and to determine whether it can elicit a second
messenger within target cells. On the other hand, the dubious origin of
the strain from which AraLAM was derived and the presence of an
analogous entity in M. smegmatis do impose searching questions
pertaining to the significance of AraLAM as a valid experimental probe
in analyzing the basis of the pathogenesis of tuberculosis.
The m/z values tabulated are based
on
We gratefully acknowledge the cooperation of Prof. A.
Malorni and S. Howe in conducting the MS/MS experiments in CNR-Napoli
(Italy); and the skillful technical assistance of Tyler Lahusen. We
thank Marilyn Hein for preparing the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
155. In addition, an inositol phosphate
tetra-arabinoside was isolated from among endoarabinase digestion
products of AraLAM and was shown to induce tumor necrosis factor-
production. Accordingly, we concluded that AraLAM is characteristic of
some rapidly growing Mycobacterium spp. It is distinct from
ManLAMs of M. tuberculosis, M. bovis BCG, and
Mycobacterium leprae not only in the absence of
mannose-capping but also in containing some terminal inositol phosphate
substituents which may account for its particular potency in inducing
macrophage activation.
(
)
is a major
component of the mycobacterial cell envelope widely implicated in the
immunopathogenesis of leprosy and tuberculosis (Brennan et
al., 1990). Both LAM and the structurally related lipomannan (LM)
from Mycobacterium tuberculosis and Mycobacterium leprae contain an intact phosphatidylinositol unit (Hunter et
al., 1986) and a branched mannan core,
[6(Man
1
2)Man
1
]
,
directly attached to position 6 of the inositol of phosphatidylinositol
(Hunter and Brennan, 1990; Chatterjee et al., 1991, 1992a,
1992b). The salient feature which distinguishes LAM from LM is the
presence of an additional immunodominant arabinan that extends from the
mannan core in an undefined manner.
Figure S1:
Scheme
1
Awareness of the subtle structural differences
among LAMs produced by various Mycobacterium sp. and their
varying capacity to modulate host immune responses prompted us to
definitively map the recognized structural motifs of LAMs from several
biologically important strains. We report here our FAB-MS and chemical
analysis of the nonreducing termini of a variety of LAMs, showing
conclusively the presence of a novel inositol phosphate capping
function associated specifically with the biologically potent AraLAM.
We also confirm that mannose capping is a common feature of LAMs from
all strains of M. tuberculosis including the H37Ra strain and
therefore may not be directly correlated with virulence.
Isolation and Purification of LAMs and LM,
Endoarabinase Digestion, and Subsequent Purification
Growth of
mycobacteria and the isolation and purification of LAMs from the
rapidly growing Mycobacterium sp., M. tuberculosis (Erdman and H37Rv), Mycobacterium leprae and
Mycobacterium smegmatis (ATCC 14468 and mc155)
were performed as described (Chatterjee et al., 1991, 1992b).
The M. smegmatis mc
155 strain is an efficient
plasmid transformation mutant and was cultured as described (Snapper
et al., 1990) for use in electroporation. LAM from H37Ra was
similarly isolated and purified from a culture given by Dr. William R.
Jacobs (Albert Einstein College of Medicine of Yeshiva University, New
York City). The selective growth of a soil microorganism,
Cellulomonas sp., on an arabinogalactan-containing medium and
the isolation and characterization of an extracellular endoarabinase
have been described (McNeil et al., 1994). Deacylated AraLAM
(5 mg) was incubated with the Cellulomonas sp. enzymes (500
µg in 1 ml), and aliquots were withdrawn after every 4 h for
reducing sugar assay (Lever, 1972). The reaction was terminated by
boiling for 2 min when the reducing sugar formation reached a plateau
after 12 h. The entire digestion mixture was applied to a column of
Bio-Gel P-6 (Bio-Rad) in water. Fractions (1 ml) were assayed for
carbohydrate (Chatterjee et al., 1993) and pooled accordingly.
Affinity Chromatography with Concanavalin A
The
voided peak, containing both Ino-P-Ara and the
mannan core of AraLAM, was reconstituted in a buffer (20 mM
Tris and 0.5 M NaCl, pH 7.4), applied to a column (1 ml) of
concanavalin A-Sepharose (Pharmacia Biotech Inc.), and eluted with 2.5
ml of the starting buffer to obtain the unbound
Ino-P-Ara
. It was further eluted with 2.5 ml of
the starting buffer containing 0.5 M
-methylmannoside in
order to recover the mannan core. The combined fractions were
immediately applied to a Sephadex G-10 column (1
117 cm,
Pharmacia) and eluted with water, and fractions prior to elution of
salt were pooled. A portion of the pooled fractions was converted to
alditol acetates and analyzed by GC to ensure the presence of arabinose
or mannose.
Perdeuteroacetylation, Mild Trifluoroacetic Acid
Hydrolysis, and Deuteroacetolysis
Perdeuteroacetylation of
intact and partially degraded LM and LAM samples was performed in
pyridine:d-acetic anhydride (1:1, v:v) at 80
°C for 2 h. LAM samples were subjected to mild acid hydrolysis with
40 mM aqueous trifluoroacetic acid (Rathburn, UK; gas phase
sequencer grade) for 20 min at 100 °C after which the reagent was
directly removed under a stream of nitrogen. For deuteroacetolysis,
perdeuteroacetylated samples were treated with a
d
-acetic anhydride:d
-acetic
acid:d
-sulfuric acid (10:10:1, v:v:v) mixture for
30-40 min at 40 °C. After quenching with water, the reaction
mixture was neutralized with a few drops of 30% aqueous ammonia, loaded
directly onto a reverse phase C18 Sep-Pak cartridge (Waters, Milford,
MA), and eluted sequentially with 2 ml of 50 and 100% aqueous
acetonitrile (Rathburn, high performance liquid chromatography grade)
after initial washes with water and 15% acetonitrile. The collected
fractions were evaporated to dryness and redissolved in methanol for
FAB-MS analysis. Similar Sep-Pak conditions were employed for further
purification of all peracetylated samples.
100-mm screw capped tube as opposed to the Mini Diazald
apparatus. To the acetylated or acetolyzed samples, diazomethane in
ether was added directly until the yellow color persisted. The reaction
mixture was left at 0 °C for 30 min and allowed to dry at room
temperature in the hood.
Permethylation, Glycosyl Composition, and Linkage
Analysis
Arabinan samples were permethylated for FAB-MS analysis
and linkage analysis by using powdered sodium hydroxide as described by
Dell et al.(1994). For analysis of inositol, samples were
hydrolyzed in 3 N HCl for 6 h at 100 °C. After evaporation
under vacuum, the hydrolysates were derivatized with the Tri-Sil®
reagent (Pierce) for about 30 min at room temperature and then
extracted into hexane. Partially methylated alditol acetates for
linkage analysis were prepared according to Albersheim et
al.(1967). GC-MS analysis was carried out on a Fisons instrument
MD800 fitted with a DB-5 capillary column (J& Scientific, Folsom,
CA). Samples were dissolved in hexane and injected on-column at 90
°C. After an initial hold at 90 °C for 1 min, the oven
temperature was increased to 290 °C at a rate of 8 °C/min for
linkage analysis. For inositol analysis, the oven temperature was
increased to 140 °C at 25 °C/min and then to 200 °C at 5
°C/min, and finally to 300 °C at 10 °C/min.
FAB-MS Analysis
FAB mass spectra were acquired
using a VG Analytical ZAB-2SE FPD mass spectrometer fitted with a
cesium ion gun operated at 20-25 kV. Data acquisition and
processing were performed using the VG Analytical Opus® software.
Monothioglycerol (Sigma) was used as matrix in both positive and
negative ion modes. CAD MS-MS collisionally activated decomposition
spectra were recorded using a Fisons VG analytical four sector ZAB-T
mass spectrometer in the array detector mode. The array was calibrated
using daughters of CsI clusters, and all experiments were carried out
at 4-kV collision cell voltage (8-kV primary ion beam) using an argon
gas pressure set to reduce the C (M + H)
of a Substance P standard to two thirds of the normal peak height
prior to gas introduction. Spectra were recorded by FAB ionization of
samples in a monothioglycerol matrix (cesium ion gun) and transmission
of the
C parent ion of interest from MS1 into the
collision cell. Array daughter spectra produced from the B/E scan of
MS2 were summed over the period of ionization of the sample.
The voided peak was deuterium
exchanged and dissolved in 600 µl of
[P NMR
H]
O. For
P NMR under
basic conditions, a drop of
N[
H]
O[
H] was
added. One-dimensional NMR spectra were recorded on a Bruker Ace-300
spectrometer (McNeil et al., 1990). H
PO
(90%) was used as the external standard.
Strategy for Deuteroacetolysis/FAB-MS Analysis of
LAMs
The selective susceptibility of the 1
6-mannosyl
linkages of the LAM core to acetolysis provided a basis for applying
time-course acetolysis/FAB-MS analysis to map the structural motifs of
various LAMs (Tsai et al., 1986). In exploratory experiments,
it was established that the mannan and the arabinan were extensively
degraded to Man
, Ara
, and Ara
in a
3-h acetolysis while the phosphatidylinositol unit was largely
preserved. To optimize the yield of putative capped and/or modified
oligosaccharides, the acetolysis reactions were monitored at intervals
by FAB-MS analysis of sample aliquots after partial separation of the
acetolyzed products on a reverse phase Sep-Pak cartridge using a
two-step gradient of 50 and 100% acetonitrile. It was anticipated that
any resulting charged species would be eluted in the earlier fraction
together with mono- and disaccharides while larger neutral
oligosaccharides would be recovered in the later fraction.
Deuteroacetolysis on perdeuteroacetylated samples was performed
throughout to avoid ambiguity in subsequent FAB-MS signal assignments.
Normal acetolysis under identical experimental conditions was also
carried out for selected samples to confirm assignments by observing
mass shifts.
(where x =
1-8)
(
)
were observed primarily as (M +
NH
)
. After more prolonged reaction,
additional molecular ion species were observed which can be assigned to
the ring open structure (I), hereafter denoted as
(+dAc
O). Since the ring open structure can be formed
at each noncleaved glycosidic linkage (II) in addition to the
reducing end, more than one dAc
O moiety may be contained in
the molecular ions as well as in the A-type ions, resulting in
multiples of 108-unit increments (Fig. S2). This phenomenon was
most pronounced with the Ara
containing fragments
(where n = 1-6)
to the extent that,
even at the early time point, they were observed almost exclusively as
dAc
O adducts, where the total number of dAc
O
moieties incorporated was usually n - 1. As a
consequence, the mass increment for the deuteroacetolyzed
Ara
series is 330 units (Ara +
dAc
O) and not 222 units (Ara).
Figure S2:
Scheme
2
Nonreducing Terminal Mannose Cap Motifs
To define
the structural difference among LAMs from a variety of sources,
deuteroacetolysis/FAB-MS analysis was performed on LAMs from M.
tuberculosis H37Ra, H37Rv, and Erdman strains, M. leprae (abbreviated as RaLAM, RvLAM, ErdLAM, and LepLAM, respectively,
for ease of description), as well as AraLAM (Chatterjee et
al., 1991). In addition, LM from M. tuberculosis Erdman
(ErdLM) was also analyzed in parallel. The positive FAB-mass spectra of
the 100% acetonitrile Sep-Pak fractions from 40-min deuteroacetolysates
of ErdLM, AraLAM, ErdLAM, RvLAM, and LepLAM in the mass range of
m/z 1190-1700 are reproduced in Fig. 1and the
assignments of the major ions are listed in . The FAB-MS
data showed that while Ara and Man
are common motifs of all LAMs, ions corresponding to
Man
Ara
(where
x` is 2 or 3, referring to the number of Man residues which
constitute the individual mannose cap) were observed as dominant
species only in the deuteroacetolysates of LAMs from the M.
tuberculosis strains. Thus LAMs from Erdman, H37Rv, and H37Ra
strains are all capped extensively with Man
and
Man
, giving Man
Ara
and
Man
Ara
, whereas AraLAM is not capped.
This observation reinforced the evidence (Prinzis et al.,
1993) that the rapidly growing strain from which AraLAM was derived
(Chatterjee et al., 1991) is not bona fide M. tuberculosis H37Ra. This strain does have many of the characteristics of M.
smegmatis including its distinct mycolic acids, although it could
not be categorically identified as such. Mannose-capping is, however,
not a phenomenon restricted to LAMs from M. tuberculosis. In
addition to LAM from M. bovis BCG, which was reported to be
mannose-capped (Prinzis et al., 1993; Venisse et al.,
1993), our FAB data (Fig. 1E) indicated that LepLAM is
also mannose-capped but apparently less so. Ions ascribed to
Man
Ara
(e.g.
m/z 1272 for Man
Ara
+
dAc
O) were evidently present but in minor abundance
relative to other Man
and Ara
ions.
Figure 1:
Positive FAB-mass spectra of the 100%
acetonitrile Sep-Pak fraction of the deuteroacetolysates of ErdLM
(A), AraLAM (B), ErdLAM (C), RvLAM
(D), and LepLAM (E), in the mass range of m/z 1180-1700. FAB-mass spectrum of the deuteroacetolysates of
RaLAM is identical to those of ErdLAM and RvLAM. Assignments of the
major ions were listed in Table I. At lower masses, A-type oxonium ions
for Ara, Ara
, Ara
, and
Man
, Man
, Man
, and molecular ions
for Ara
, Ara
, and Man
, Man
were afforded by all LAM samples while those of
Man
Ara
were present in all LAMs except AraLAM.
Above m/z 1700, the Man series extended to Man
and
Man
, with a much weaker signal for
Man
.
These data were further corroborated by methylation
analysis of RvLAM, RaLAM, and LepLAM (). In contrast to
AraLAM, which yielded almost equal amounts of terminal Araf and 2-linked Araf but no 2-linked Manp, both
RvLAM and RaLAM gave terminal Araf and 2-linked Araf in about a 1:2 ratio, as well as substantial amounts of 2-linked
Manp. Since each 2-linked Araf should be substituted
with a terminal Araf unless it is further capped with
Manp1
(2-Manp
1
)
,
where x in this case is 0-3, the methylation analysis
data therefore suggested that approximately half of the nonreducing
terminal Araf of both RaLAM and RvLAM are capped with Man
residues. The approximate 1:1 ratio of terminal Araf:2-linked
Araf of LepLAM indicated that most of its Araf termini are not capped. However, the presence of some degree of
mannose capping in LepLAM was evident from both the FAB-MS data and the
presence of significant amounts of 2-linked Man. Assuming that the
dominant cap is Manp1
2Manp and that all
2-linked Manp are constituents of the cap, the degree of
mannose capping in LepLAM was estimated to be not more than 30%,
considerably lower than RvLAM, RaLAM, and ErdLAM (
70%).
Presence of Additional Structural Motifs
The
deuteroacetolysis/FAB-MS data also attested to the presence of a common
branched mannan core
[6(Man
1
2)Man
1
]
,
the
1
6 bonds of which were acetolyzed much more readily than
the
1
2 linkages. Thus, signals corresponding to
Man
, Man
, Man
, and Man
were much stronger relative to those corresponding to
Man
, Man
, and Man
in all LAMs
examined. LM possessed only the Man
series (x = 1-8). In the 50% acetonitrile Sep-Pak fraction, a
trace amount of Ara
was present in the FAB-mass spectrum of
deuteroacetolyzed ErdLM, the origin of which is not clear. Otherwise,
the only other prominent signals present were the molecular ions and
A-type fragment ions of Man
and Man
. In
contrast, the FAB-mass spectra of the deuteroacetolysates of all LAMs
were dominated by Ara
and Ara
in addition to
Man
and Man
. The positive FAB-mass spectra of
the 50% acetonitrile Sep-Pak fractions of the deuteroacetolysates of
ErdLM, AraLAM, and ErdLAM in the mass range of m/z 650 to 1180
(Fig. 2) contained ions at m/z 720 and 725,
corresponding, respectively, to (M + NH
)
and (M + Na)
of Man
. Their
under-deuteroacetylated counterparts afforded the signals at m/z 675 and 680. In addition, Ara
was observed as (M
+ dAc
O + NH
)
and (M
+ dAc
O + Na)
at m/z 678
and 683, respectively.
Figure 2:
Positive FAB-mass spectra of the 50%
acetonitrile Sep-Pak fraction of the deuteroacetolysates of ErdLM
(A), AraLAM (B), and ErdLAM (C), in the mass
range of m/z 650 to 1180. All signals at lower masses were
attributed to Ara, Ara
, Man
, and
Man
. The signal at m/z 690 in B corresponds to the oxonium ion of Ino-P-Ara
.
Signals at m/z 977 and 902 in C correspond to (M
+ Na)
of under-deuteroacetylated Man
and Man
Ara
, respectively. Other signals
at 45 units lower than those assigned correspond to
under-deuteroacetylated species.
Evidence for a Novel Ino-P-Ara
Strikingly, the FAB-mass spectrum of the
deuteroacetolysates of AraLAM (Fig. 2B) was dominated by
two signals not present in the corresponding spectra of all other
LAM/LM samples analyzed. The mass difference of 330 units (222 +
108) between the signals at m/z 775 and m/z 1105 was
indicative of an X-Ara Motif
in AraLAM
series with the
latter containing an additional Ara (222 units) residue and a
dAc
O adduct (108 units). In the negative ion mode, the same
sample afforded two correspondingly prominent negative ion signals at
m/z 751 and 1081, which were not found in the spectra of any
other LAM. It thus appeared that a moiety of
X-Ara
yielded strong (M -
H)
ions in the negative ion mode while desorbing
primarily as (M + Na)
and (M - H +
2Na)
in the positive ion mode
(Fig. 2B). When the deuteroacetolyzed AraLAM was treated
with diazomethane, the relevant positive ions were shifted 14 units
higher while the negative ions were abolished, suggesting that the
negative charge could be due to the presence of a phosphodiester which
was methylated by diazomethane and hence lost its charge. Taken
together, these data were consistent with Ino-P-Ara
and
[Ino-P-Ara
(+dAc
O)] as the two
components unique to the deuteroacetolysates of AraLAM.
Ara
and
Ara
but not Man
were
observed, indicating that only the arabinofuranosyl linkages were
partially hydrolyzed. Specifically, RaLAM afforded major (M +
Na)
molecular ion signals at m/z 947, 1169,
1391, 1613 (Man
Ara
,
Man
Ara
, Man
Ara
,
Man
Ara
, respectively) and m/z 1244,
1466, 1688 (Man
Ara
,
Man
Ara
, Man
Ara
,
respectively), in addition to m/z 353, 575, 797, 1019
(Ara
, Ara
, Ara
, Ara
,
respectively), whereas AraLAM gave only the Ara
series (data not shown).
of Ino-P-Ara
. At 222
units higher (m/z 973) was the (M - H)
of Ino-P-Ara
which, as expected, lacked the extra
dAc
O moiety found in the deuteroacetolyzed sample whose
corresponding ion occurred at m/z 1081. In addition,
Ino-P-Ara
were also produced, affording a series of
ions with mass intervals of 222 units. Upon complete hydrolysis of this
sample, inositol was identified as its trimethylsilyl derivative in
subsequent GC-electron impact-MS analysis (characteristic ions at
m/z 318, 305, 217, 147, 73) where it eluted at a retention
time identical to the trimethylsilyl derivative of an authentic
myo-inositol standard. Information on the attachment point of
the phosphodiester linkage was obtained by negative ion CAD MS-MS of
the molecular ion for Ino-P-Ara
at m/z 751
(Fig. 3, inset). A prominent fragment ion at m/z 627 was obtained, consistent with loss of deuteroacetyl
substituents from positions 1 and 2 of the reducing end Ara via an
established fragmentation pathway (Dell et al., 1990).
Analogous fragment ions were observed in the CAD spectra of the Ara
oligomers (see Fig. 4), suggesting that the phosphodiester moiety
is not attached at position 2.
Figure 3:
The negative FAB-mass spectrum of
perdeuteroacetylated mild trifluoroacetic acid hydrolysates of AraLAM
after Sep-Pak purification. The negatively charged Ino-P-Ara were
eluted with 50% aqueous acetonitrile while the nonhydrolyzed mannan
core was retained on the C18 cartridge. Signals observed correspond to
(M - H) of Ino-P-Ara
through to
Ino-P-Ara
, each accompanied by under-deuteroacetylated
species. Inset, negative ion CAD MS-MS spectrum of the
molecular ion at m/z 751. Signals at m/z 706 and 688
correspond to loss of a trideuteroacetyl and a trideuteroacetic acid
moiety respectively. Other signals assigned to fragment ions resulting
from collisional activation of m/z 751 are:
H
PO
(m/z 97);
(P-Ara) (m/z 364); (Ino-P)
(m/z 484); and the ion at m/z 627 described in
the text.
Figure 4:
Positive ion CAD mass spectra of
Ara after collisional activation of the (M +
Na)
molecular ion at m/z 1019 (A)
and Ara
after collisional activation of the (M +
Na)
molecular ion at m/z 1463 (B).
Sequence and/or linkage informative fragment ions were assigned as
shown schematically on the figures. Other secondary fragment ions were
derived from loss of a trideuteroacetic acid moiety (minus 63 units) or
multiple cleavages.
Localizing the Ino-P-Ara
To further define the location of the
Ino-P-Ara Motifs
structural motif and to ascertain
whether it carries any other additional acid-labile moiety which might
have been hydrolyzed or deuteroacetolyzed, deacylated AraLAM was
digested with the Cellulomonas endoarabinase. This enzyme was
previously shown to be highly selective for the 5-linked Araf residues which follow a branched 3,5-linked Araf unit and
the fourth Araf residue from the nonreducing end in the case
of the linear terminal motif (Chatterjee et al., 1993; McNeil
et al., 1994). The endoarabinase-digested AraLAM was
fractionated on a Bio-Gel P-6 column, pooled into void and fractions A,
B, and C, and screened by FAB-MS after perdeuteroacetylation and
permethylation (I). The dominant components in fractions
A, B, and C were Ara
, Ara
, and Ara
,
respectively. In agreement with the known enzyme specificity, the
Ara
and Ara
digestion products were shown to be
branched and linear, respectively, by methylation analysis
(). This was further confirmed by tandem mass
spectrometric analysis. The CAD spectrum of Ara
[m/z 1019 for (M + Na)
]
afforded sodiated reducing terminal ring cleavage ions at m/z 336, 558, and 780, corresponding to Ara
,
Ara
, and Ara
, respectively
(Fig. 4A). More importantly, glycosidic cleavage
concomitant with elimination of the 2-O-substituent yielded
the nonreducing terminal cleavage ions at m/z 895
(Ara
) and 673 (Ara
) but not 451
(Ara
), thus confirming the known Ara1
2Ara linkage at
the nonreducing end of the linear Ara
chain.
at m/z 1463 afforded analogous fragment ions
(Fig. 4B). Significantly, sodiated ring cleavage ions
were observed only for Ara
, Ara
, and Ara
at m/z 336, 1002, and 1224, respectively, and not for
Ara
or Ara
. In addition, strong nonreducing
terminal ions resulting from glycosidic cleavage concomitant with
elimination of the 2-O-substituent were observed only for
Ara
(at m/z 1117) and Ara
(at m/z 1339). The CAD spectrum afforded by Ara
therefore
firmly demonstrated that the major Ara
component produced
by endoarabinase digestion corresponded to the previously characterized
branched structure.
motifs
coeluted with the mannan core in the voided peak, presumably due to the
negative charge they carried. The presence of two different families of
phosphodiester-containing oligomers in the voided peak was further
supported by
P NMR experiments. In contrast to the
spectrum afforded by the mannan core similarly isolated from ErdLAM,
which contained only a single phosphodiester signal at
0.68 at
neutral pH, the NMR spectrum afforded by the void fraction from AraLAM
showed two distinct signals of phosphodiester at
0.71 and 0.45
ppm. At basic pH, the chemical shifts of the phosphodiester remained
unchanged, indicating that both the phosphates were in the form of
phosphodiester, one belonging to the Man-Ino-P-glycerol from the mannan
core (
0.71) and the other attributable to
Ino-P-Ara
.
molecular ion signals were present at m/z 1417, 1639,
1862, 2084, and 2306, corresponding to Ino-P-Ara
,
Ino-P-Ara
, Ino-P-Ara
, Ino-P-Ara
,
and Ino-P-Ara
, respectively (Fig. 5). Interestingly,
although the larger Ino-P-Ara
were also produced,
these were clearly very minor components relative to the predominant
Ino-P-Ara
. In the positive ion mode, the Ino-P-Ara
series afforded both (M + NH
)
and (M + Na)
molecular ions (data not
shown). Significantly, despite the intense molecular ion signals of
Ino-P-Ara
, A-type oxonium ions of Ara
and
Ara
were not prominent among the matrix noise. Instead, a
strong ion was present at m/z 690, corresponding to the A-type
ion Ino-P-Ara
. Following diazomethane
treatment, this ion shifted to m/z 704. Similarly, all
molecular ions were shifted 14 units higher, consistent with
monomethylation of a phosphodiester moiety. In addition, the permethyl
derivatives of Ino-P-Ara
afforded a major (M
+ NH
)
molecular ion at m/z 1016 and a strong A-type ion at m/z 487, corresponding to
Ino-P-Ara
and Ino-P-Ara
,
respectively, with all hydroxyl groups including that of the
phosphodiester moiety being fully methylated. Taken together, the data
were fully consistent with a novel inositol phosphate moiety capping a
portion of the nonreducing termini of the linear arabinan chain which,
upon digestion with endoarabinase, yielded predominantly
Ino-P-Ara
.
Figure 5:
The negative FAB-mass spectrum of
perdeuteroacetylated Ino-P-Ara recovered from the endoarabinase
digestion products of AraLAM. Signals at 45 units lower than those
assigned as (M - H) of Ino-P-Ara correspond to
under-deuteroacetylated species. Inset, schematic
representation of the provisional structure for Ino-P-Ara
which afforded the ion at m/z 1417. The glycosyl linkage
positions for the arabinan were not defined. Linkage analysis of the
purified Ino-P-Ara sample gave only 2-linked and 5-linked
Araf. The absence of terminal Ara is consistent with the Ino-P
being attached to the nonreducing arabinose. The attachment point for
the phosphate is likely to be position 5 in view of the CAD MS/MS
experiment which indicated that deuteroacetylated Ino-P-Ara
has a deuteroacetyl substituent at position 2. However,
attachment to position 3 cannot be ruled
out.
LAMs from M. smegmatis
The identification of the
Ino-P-Ara motif on AraLAM from an unknown, rapidly
growing Mycobacterium sp. prompted us to examine LAMs from the
speciated, fast growing M. smegmatis (ATCC 14468 and
mc
155). Methylation analysis indicated that they are not
mannose-capped (). After deuteroacetolysis, the same
positive and negative ions assigned to Ino-P-Ara
(m/z 775 and 751, respectively) and [Ino-P-Ara
(+dAc
O)] (m/z 1105 and 1081,
respectively) were produced. Mild trifluoroacetic acid hydrolysis
followed by perdeuteroacetylation and C18 Sep-pak separation yielded
the same series of negative ions attributed to
Ino-P-Ara
. Finally, endoarabinase digestion of LAM
from the mc
155 strain followed by Bio-Gel P-6
fractionation, perdeuteroacetylation, and FAB-MS screening afforded the
same m/z 1417 ion ((M - H)
of
Ino-P-Ara
) as the dominant negative ion in the void
fraction. Although rigorous biochemical characterization was not
performed, the FAB-MS data strongly indicated that the same inositol
phosphate capping motif as that characterized for AraLAM is also
present on LAM from M. smegmatis.
Biological Activity of Ino-P-Ara
To obtain the Ino-P-Ara from
AraLAM
components
produced by endoarabinase digestion of AraLAM for biological studies,
the voided peak from Bio-Gel P-6 column was passed through a
concanavalin A-Sepharose affinity column to remove the mannan core. The
potency of Ino-P-Ara
in eliciting TNF-
production in murine bone marrow-derived macrophages was compared to
that of intact AraLAM and lipopolysaccharide. The assay involved
reverse transcription of stimulus-induced mRNA for TNF-
, followed
by PCR amplification of its specific sequences (Wynn et al.,
1993). It was found that the potency of AraLAM at 1 µg was 30%
lower than that of an equal amount of lipopolysaccharide, whereas that
of Ino-P-Ara
was 20% higher (data not shown). The
inositol phosphate-capped Ara termini in AraLAM may thus be responsible
for its known potency in elevated level of TNF-
induction
(Chatterjee et al., 1992c).
O ring open forms, FAB-MS analysis of the
acetolysates or deuteroacetolysates of LAMs has the advantage of
simultaneously defining both nonreducing termini of the differentially
capped arabinan and the mannan core. In a time course experiment, an
early time point can be chosen to optimize the recovery of
Ara-containing fragments and limit the acetolysis of the mannan core
specifically to Man
1
6Man linkages while more extended
acetolysis affords fragments of the mannosyl phosphatidylinositol
anchor (Khoo et al., 1995). Any additional acyl or
glycopyranosyl substituent(s) on the arabinan and mannan core will be
uncovered by this approach. Indeed, we have preliminary evidence for
the presence of additional negatively charged substituents on the core,
the identity of which is currently under investigation.
-Araf residue, replacing the Man
or Man
unit that typifies ManLAM. Incidentally, this is also the point
of attachment of the mycolyl substituent in the
arabinogalactan-peptidoglycan complex of mycobacterial cell walls
(McNeil et al., 1991).
motif was
identified in its acid hydrolysates, deuteroacetolysates, and
endoarabinase digestion products. Although the rapidly growing strain
which produces AraLAM was originally believed to be an aberrant M.
tuberculosis H37Ra, it now seems possible that it is M.
smegmatis. Alternatively, it may have acquired these
characteristics of M. smegmatis or rapidly growing species
during subculturing through preferential growth of a contaminating
rapid growing species. A mix-up in cultures may also have happened.
response (Chatterjee et al.,
1992c) were recently extended to examination of the expression of early
response genes in murine bone marrow-derived macrophages (Roach et
al., 1993). Both AraLAM and ManLAM elicited immediate early gene
(c-fos, JE, KC) responses. However, only
AraLAM, like lipopolysaccharide, induced both TNF-
secretion and a
potentially lethal TNF-dependent-NO response (Roach et al.,
1995). From these studies, it appeared that LAMs from strains of M.
tuberculosis, through mannose-capping, are designed to avoid
stimulation of macrophage early gene expression which will lead to the
induction of a potentially lethal TNF-dependent NO response. However,
the present finding that LAM from avirulent M. tuberculosis H37Ra as well as that from the vaccine strain M. bovis BCG (Prinzis et al., 1993; Venisse et al., 1993)
are both mannose-capped indicates that any putative role for LAM in the
variable virulence of the strains of the M. tuberculosis complex is related to factor(s) other than mannose capping. It has
recently been shown that mannose-capping on LAM may be involved in the
uptake of mycobacteria by direct binding to the mannose receptors of
macrophages (Schlesinger, 1993; Schlesinger et al., 1994).
Since all M. tuberculosis strains produce mannose-capped LAM,
it is possible that the mannose caps are essential for their initial
binding to macrophages. Virulence and pathogenesis of strains would
then be dependent on events subsequent to this binding.
Table:
Positive
ions observed in the FAB-mass spectra of the 100% acetonitrile Sep-Pak
fraction of the deuteroacetolysates of LAMs in the mass range of m/z
1180-1700 (Fig. 1)
C-monoisotopic nominal masses calculated while those
labeled in Fig. 1 are computer-assigned accurate masses, rounded out to
the nearest integer.
Table: 0p4in
From Chatterjee et al. (1991).(119)
Table:
Major
components identified by FAB-MS screening of perdeuteroacetylated
samples from the pooled Bio-Gel P-6 fractions of endoarabinase digested
AraLAM
Table:
Methylation analysis of the pooled Bio-Gel P-6
fractions of endoarabinase digested AraLAM
, Ara
, Ara
,
etc., a single arabinose, an arabinobiose, an arabinotriose, etc.;
Manp, mannopyranose; Man
, Man
,
Man
, etc., a single mannose, a mannobiose, a mannotriose,
etc.; LM, lipomannan; AraLAM, LAM from the rapidly growing
Mycobacterium sp.; ErdLM and ErdLAM, LM and LAM from M.
tuberculosis Erdman; RvLAM and RaLAM, LAM from M. tuberculosis H37Rv and H37Ra; CAD, collision-activated dissociation; FAB, fast
atom bombardment; GC, gas chromatography; Ino-P, inositol phosphate;
MS, mass spectrometry; TNF, tumor necrosis factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.