Glycobiology vol. 8 no. 10 pp.

955962, 1998

Isolation and characterization of the major form of polyprenyl-phospho-mannose

from Mycobacterium smegmatis

Beata A.Wolucka2 and Edmond de Hoffmann1

Department of Applied Chemistry and Bioindustries and 1Department of
Chemistry, University of Louvain, Louvain, Belgium
Received on September 2, 1997; revised on March 21, 1998; accepted on
March 31, 1998
2To

whom correspondence should be addressed at: Laboratory of Brewing

Science, Department of Applied Chemistry and Bioindustries, University of
Louvain, Pl. Croix du Sud 2/7, B-1348 Louvain-la-Neuve, Belgium

Key words: cell-wall/mannolipid/mycobacteria/polyprenol/

tandem mass spectrometry

Introduction
The cell-wall of mycobacteria comprises a covalently linked
peptidoglycan-arabinogalactan-mycolate framework and numerous other components of miscellaneous nature, such as lipids and
glycolipids, proteins, membrane-bound or free lipomannans/
mannans and lipoarabinomannans/arabinomannans, disposed
within the wall in an ill-defined manner. Because of the
uniqueness of this structure and the fact that some key antituberculosis drugs act at the level of the cell-wall, a lot of research
has been undertaken to elucidate the underlying biosynthetic
pathways in order to unveil the mechanisms of drug resistance in
mycobacteria and to develop new antimycobacterial agents.
1998 Oxford University Press

955

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We isolated from the endogenous polyprenyl-phospho-sugar

pool of Mycobacterium smegmatis two mannose-containing
compounds, i.e., a partially saturated C35-octahydroheptaprenyl-P-mannose and a fully unsaturated C50-decaprenyl-P-mannose. The relative amount of C35-polyprenyl-P-mannose in mycobacterial cells was comparable to that of decaprenylP-pentoses and, at least, an order of magnitude higher than that
of C50-decaprenyl-P-mannose. The major form of mycobacterial polyprenyl-P-mannose was structurally characterized by
combined gas chromatography-mass spectrometry, fast-atom
bombardment tandem mass spectrometry and proton-nuclear
magnetic resonance spectroscopy as -D-mannopyranosylmonophospho-(C35)octahydroheptaprenol of which all three
isoprene units have Z (cis) configuration. The differences in the
structure and cellular concentrations of the mycobacterial mannosyl-P-polyprenols reflect distinct biochemical pathways of the
two compounds and suggest the existence of specific GDPMan:polyprenyl-P mannosyltransferases (synthetases) able to
distinguish between C35-octahydroheptaprenyl- and C50-decaprenyl- phosphates of mycobacteria. Since the 6-O-mycoloylated form of C35-octahydroheptaprenyl-P-mannose isolated
from M. smegmatis is apparently involved in mycolate rather
than mannosyl transfer reactions, we speculate that a catabolic
pathway responsible for degradation of C35-P-mannose and recycling C35-octahydroheptaprenyl phosphate might exist in mycobacteria.

During the last years, the biosynthesis of D-arabinofuranosyl

residues of the cell-wall arabinogalactans and arabinomannans
has been at the center of interest. The discovery of an important
group of polyprenyl-P-sugars containing arabinose, ribose, and
mannose in Mycobacterium smegmatis (Wolucka, 1992)
followed by the complete structural characterization of the
decaprenyl-P-arabinose component (Wolucka et al., 1994) was
imperative for the subsequent chemical synthesis of a substrate
for the mycobacterial arabinofuranosyltransferases and development of the enzyme assay (Lee et al., 1995). The observation that
ethambutol treatment results in accumulation of decaprenylP-arabinose in mycobacteria pointed to arabinotransferases as
target for the drug (Wolucka et al., 1994). This has been
confirmed by the studies with ethambutol-resistant mutants of
M.tuberculosis (Telenti et al., 1997) and M.avium (Belanger et al.,
1996) which demonstrated that the overexpression of decaprenylP-arabinose-specific arabinosyltransferases leads to ethambutol
resistance in mycobacteria.
Another important issue in studies on the D-arabinose origin is
the discovery of decaprenyl-P-ribose in mycobacteria (Wolucka
and de Hoffmann, 1995). Although the function of this compound
remains unknown, its existence and an apparent biochemical
relationship to the 2-epimer, decaprenyl-P-arabinose, due to a
hypothetical 2-epimerase activity (Wolucka et al., 1994) were
the first hints to the role of activated ribofuranosyl derivatives in
the biosynthesis of mycobacterial walls. Further studies of
Scherman et al. (1996) demonstrated that 5-phosphoribosyl
pyrophosphate, a common precursor for the biosynthesis of
nucleic acids, serves as a donor of pentosyl for both decaprenyl-P
derivatives, decaprenyl-P-arabinose and decaprenyl-P-ribose,
through a polyprenyl-P-pentosyl 5-phosphate intermediate. The
level at which the 2-epimerization takes place (pRpp or
polyprenyl-P-ribosyl 5-phosphate) has not been yet defined.
Contrary to polyprenyl-P-pentoses, the polyprenyl-P-mannose
derivatives have received only little attention. The fact that
incubation of mycobacterial membranes with GDP-mannose leads
to the formation of two different mannosyl-P-polyprenols, i.e.,
mannosyl-P-decaprenol and mannosyl-P-octahydroheptaprenol,
was reported many years ago (Takayama and Goldman, 1970;
Takayama et al., 1973). However, the endogenous mannosylP-polyprenols and the enzymes responsible for their synthesis have
not been characterized.
Mycobacterial cell envelopes comprise many mannose-containing molecules, the major fraction of which represent phosphatidyl-myo-inositol-oligomannosides (PIMx, where x = 26)
and the structurally related (lipo)mannans and (lipo)arabinomannans (Besra and Chatterjee, 1994). The common feature of these
compounds is the presence of a linear chain of (16)-linked
mannose backbone with (12)-linked mannosyl side chains.
Certain mycobacteria produce also species-specific lipooligosaccharides (McNeil et al., 1987) and phenolic glycolipids (Rivire
et al., 1987) which contain mannose. Another type of mannosecontaining molecule is an (14)-linked polymer of 3-O-methyl-

B.A.Wolucka and E.de Hoffmann

Results
Isolation and purification of mannosyl-P-polyprenols of
Mycobacterium smegmatis
Endogenous polyprenyl-P-sugars were isolated from M.smegmatis
cells by a two-step procedure consisting of extraction with hot
ethanol, followed by partitioning of the dried extract into a biphasic
mixture of chloroform/methanol/water (8:4:3). DEAE-cellulose
chromatography of the lipidic material followed by alkaline
hydrolysis yielded a fraction of alkali-stable polyprenyl-P-sugars,
which were resolved further by a preparative TLC into four major
bands (Rf 0.36 to 0.46) (Wolucka, 1992; Wolucka et al., 1994). The
fast migrating compounds (Rf 0.46 and 0.43) were shown to be
decaprenyl-P-arabinose
(-D-arabinofuranosyl-monophospho(C50)decaprenol) (Wolucka et al., 1994) and a mixture of
decaprenyl-P-ribose (-D-ribofuranosyl-monophospho-(C50)decaprenol) and C35-octahydroheptaprenyl-P-arabinose (-D-arabinosyl-monophospho-(C35)octahydroheptaprenol) (Wolucka and
de Hoffmann, 1995), respectively. Similar to the fast-migrating
polyprenyl-P-pentoses, the slowly migrating bands (Rf 0.39 and
0.36) gave a glycolipid-positive reaction with -naphthol but upon
mild acid hydrolysis released mannose, indicating to polyprenylP-mannose derivatives. When rechromatographed, the purified
compounds (Rf 0.39 and 0.36) migrated on silica TLC in a similar
way and gave only one spot which was J2- and -naphthol positive.
Since free polyprenyl phosphates migrate faster in this system (Rf
0.53 and 0.50 for C50-P and C35-P, respectively), we can deduce
therefore that the purified compounds did not contain polyprenyl
phosphates in a free form.
956

Fig. 1. Gas chromatogram of trimethylsilyl ethers of methyl glycosides

derived from the purified C35-polyprenyl-P-mannose.
C35-polyprenyl-P-mannose was subjected to acid methanolysis followed by
trimethylsilylation and injected to GC-MS. IS, perseitol (internal standard).

Sugar composition
Methanolysis of the intact mannolipids, followed by trimethylsilylation and GC-MS of the resulting TMS-ethers of methyl
glycosides, revealed the presence of only mannose. Figure 1 shows
the total ion chromatogram obtained for the sample derived from
the slowest-migrating mannolipid (Rf 0.36), with two peaks, the
retention times and mass spectra (m/z 467, 435, 377, 361, 345, 303,
287, 271) of which corresponded to the anomers of trimethylsilylated methyl-mannopyranoside (Kakehi and Honda, 1989).
Quantification of sugar in both mannolipid samples demonstrated
that the slowest-migrating compound (Rf 0.36) contained 20 times
more mannose than the other mannolipid (Rf 0.39). Therefore, the
mannolipid of Rf 0.36 (C35-P-mannose; see below) is the major
form of polyprenyl-P-mannose in M.smegmatis.
Fast-atom bombardment tandem mass spectrometry of
mannosyl-P-polyprenols
Both mannolipids produced, by negative-ion fast-atom bombardment mass spectrometry, an abundant ion of the deprotonated
molecule at m/z 743 (Figure 2A) and m/z 939 (not shown) for the
compounds of Rf 0.36 and 0.39, respectively. The spectra showed
also the presence of a fragment ion derived from the loss of
mannosyl residue (-162 a.m.u.) and corresponding to polyprenylP at m/z 581 (Figure 2A) and m/z 777 (not shown) for the
compound of Rf 0.36 and 0.39, respectively. Thus, it could be
deduced that the slowest-migrating mannolipid (MW = 744)
consisted of C35-octahydroheptaprenyl-P-mannose, whereas the
other compound (Rf 0.39) was C50-decaprenyl-P-mannose.
In Figure 2A, the deprotonated molecule of C35-P-mannose
(m/z 743) and the C35-P fragment (m/z 581) are accompanied by
ions (+16 and +32 a.m.u.) corresponding to oxidation products at
m/z 759, 775 and m/z 597, 613, respectively. The oxidation
process took place, probably, in the FAB source and was inherent
to the FAB ionization and not to the sample, since no evidence of
oxidated species could be obtained by a subsequent 1H-NMR
analysis of the same sample (see below). Additionally, in the FAB
spectrum, signals of adducts with a thioglycerol molecule of the
matrix (+108 a.m.u.) can be observed for each of the above-described ions, the presence of which renders the spectrum even
more complex.
Further structural information was obtained by tandem mass
spectrometry of the deprotonated molecule of each mannolipid

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mannose of M.smegmatis (Gray and Ballou, 1971). In addition,

mannose was identified as the principal sugar component of the
recently isolated mycobacterial glycoproteins (Dobos et al.,
1996; Herrmann et al., 1996).
Consequently, mannosyl-P-polyprenols were regarded as
possible mannosyl donors in the formation of the mannose-containing cell-envelope components of mycobacteria. Thus, Yokoyama and Ballou (1989) demonstrated that decaprenylP-[14C]mannose serves as substrate for the in vitro synthesis of
free (16) oligomannosides and, recently, Besra et al. (1997)
reported that a linear (16) mannooligosaccharide attached to
phosphatidylinositol (a linear lipomannan) is formed at the
expense of polyisoprenyl-P-mannose by mycobacterial membranes. However, the fact of the existence of 6-O-mycoloyl esters
of octahydroheptaprenyl-P-mannose in M.smegmatis (Besra
et al., 1994) does not fit to this classic view about the role of
polyprenyl-P-mannose in mycobacteria and, unexpectedly, links
the octahydroheptaprenyl-P derivative to the metabolism of the
cell-wall mycolic acids.
In the present work, we describe the isolation and characterization of endogenous mannosyl-P-polyprenols of M.smegmatis. In
particular, it is shown that octahydroheptaprenyl-P-mannose is
the major form of the mycobacterial polyprenyl-P-mannose.
Finally, a possible existence of distinct polyprenyl-P-mannose
synthases specific for each phospholipid and of an enzymatic
system involved in the catabolism of octahydroheptaprenylP-mannose and octahydroheptaprenyl-P recycling in mycobacteria will be discussed.

PolyprenylPmannose of mycobacteria

Fig. 2. Negative-ion fast-atom bombardment tandem mass spectrometry

of the purified C35-polyprenyl-P-mannose of M. smegmatis. (A) the
FAB-MS spectrum; (B) the product ion spectrum of the m/z 743 ion. 108
atomic mass units corresponds to a thioglycerol molecule of the matrix.

(Figures 2B, 3). The product ion spectrum of the [M - H] ion of

the mannosyl-P-polyprenols contained a signal of the polyprenyl
phosphate ion, i.e., C35-octahydroheptaprenyl-P at m/z 581 in the
case of C35-P-mannose, and C50-decaprenyl-P at m/z 777 for
C50-P-mannose. In addition, a dehydration product (at m/z 725
and 921) and a characteristic [polyprenyl-PO4-(C2H3O)] fragment at m/z 623 and m/z 819 could be observed in the tandem
mass spectra of the C35- (Figure 2B) and C50- (Figure 3)
derivatives, respectively. The presence in the product ion
spectrum of an abundant [polyprenyl-PO4-(C2H3O)] fragment
derived from cleavage across the sugar ring is typical for
polyprenyl-P-sugars with cis-disposition of the O-1 and O-2
oxygens of the glycosyl residue (Wolucka et al., 1996b). In the
case of D-mannose-containing compounds, the cis configuration
of the phosphate and 2-hydroxyl groups of the mannosyl residue
implies that mannose is -linked to the C35-octahydroheptaprenyl- and C50-decaprenyl- phosphates.

Fig. 4. The 500 MHz 1H-NMR spectrum of the C35-polyprenyl-P-mannose.

The sample was obtained from 120 g of M.smegmatis cells. The purified
C35-polyprenyl-P-mannose (121 nmol based on sugar estimation) was
dissolved in a mixture of deacidified C[2H]Cl3 (330 l) and C[2H]3O[2H]
(120 l).

The proton-NMR analysis of

C35-octahydroheptaprenyl-P-mannose
The 1H-NMR spectrum of C35-P-mannose (Figure 4) revealed
the presence of the anomeric proton signal at 5.005 ppm
characteristic of -mannosyl. The resonances at the region
between chemical shifts 3.3 and 4.1 could be assigned to the ring
protons of mannopyranosyl according to OConnor et al. (1979).
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Fig. 3. Negative-ion fast-atom bombardment tandem mass spectrometry of

the purified C50-decaprenyl-P-mannose of M.smegmatis. The product ion
spectrum of the [M - H] deprotonated molecule (m/z 939).

B.A.Wolucka and E.de Hoffmann

Fig. 5. The proposed structure of the C35-polyprenyl-P-mannose,

-D-mannopyranosyl-1-monophospho-octahydroheptaprenol, of
Mycobacterium smegmatis.

958

Table I. Estimation of mild acid-labile mannose in the DEAE-cellulose

fractions containing the mycobacterial [14C]polyprenyl-P-sugars before and
after mild alkaline hydrolysis
DEAE-cellulose fraction
eluted with 30 mM HCOONH4

c.p.m. in mannose
liberated to the water-phase by mild acid

Before alkali treatment

5025

After mild-alkali treatment

4613

M.smegmatis cells were labeled with [14C]glucose and the lipid extract was
chromatographed on a DEAE-cellulose column, as described in Materials
and methods. Aliquots (8000 c.p.m.; in duplicates) of the 30 mM ammonium
formate fraction which contained [14C]polyprenyl-P-sugars, were evaporated
to dryness and submitted or not to a mild-alkaline hydrolysis (0.1 N NaOH in
ethanol at 37
C for 40 min) followed by partitioning into
chloroform/methanol/water (8: 4:3). The aqueous phases were discarded and
the organic phases were evaporated, hydrolyzed in 10 mM HCl for 5 min at
100
C, and partitioned again in chloroform/methanol/water (8:4:3). The
sugars released to the water-phase were resolved by a TLC on MN300
cellulose. After autoradiography, the radioactive spots comigrating with a
mannose standard were scraped off and counted.

Table II gives a summary of the quantitative data (based on

sugar estimation) on the composition of the endogenous polyprenyl-P-sugar pool of M.smegmatis. C50-Decaprenyl-P-ribose is
the major component (340 nmol/100 g of wet weight), followed
by C50-decaprenyl-P-arabinose (86 nmol/100 g of wet weight)
and C35-octahydroheptaprenyl-P-mannose (103 nmol/100 g of
wet weight), whereas C35-octahydroheptaprenyl-P-arabinose
and C50-decaprenyl-P-mannose are present in much lower
amounts (27 and 5 nmol/100 g wet weight, respectively) in the
cell. Thus, only about 5% of the total polyprenyl-P-mannose is
present under the form of decaprenyl-P derivative and the other
95% of mannosyl residues is associated with the shorter, partially
saturated C35-octahydroheptaprenyl-P (Table II). These structural and quantitative differences (Table II) point to distinct
biological functions of the polyprenyl-P-sugars in mycobacteria,
some of which we begin to understand, as those of decaprenylP-arabinose (Belanger et al., 1996) and C35-P-mannose (Besra et
al., 1994), but functions of the other compounds (decaprenylP-ribose and octahydroheptaprenyl-P-arabinose) still await an
elucidation.

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The downfield resonances at chemical shifts 5.34 and 5.05

resulted from the methine protons of -C(CH3)=CH-CH2-O-P and
-C(CH3)=CH-, respectively, whereas the signal at 4.38 ppm
corresponded to methylene protons of =CH-CH2-OP of the
C35-octahydroheptaprenyl-P moiety. In the upfield region, the
resonances observed at 0.810.83 and 1.2 can be assigned to the
protons of methyl and methylene groups of the partially saturated
lipid chain, respectively.
The proton-NMR spectrum of C35-P-mannose gives also a
valuable information about the stereochemistry of the isoprene
units of the mycobacterial octahydroheptaprenyl-P. According to
our previous data (Wolucka et al., 1994, 1996a; Wolucka and de
Hoffmann, 1995), the signal observed at 1.69 ppm can be
assigned to cis-methyl protons of the unsaturated -isoprene
residue of octahydroheptaprenyl-P, and the resonance at 1.63 to
cis-methyls of the internal Z isoprene units (Figure 4). The lack
of signal at 1.57 ppm characteristic of isoprenyl trans-methyl
groups in the 1H-NMR spectrum of C35-octahydroheptaprenylP-mannose indicates clearly that the mycobacterial octahydroheptaprenol does not contain any E (trans) isoprene residues and,
consequently, that all three isoprene units of the lipid have Z (cis)
configuration. This is in contradiction with the structure of the
octahydroheptaprenyl residue with one E (trans) isoprene unit as
proposed by Besra et al. (1994), who wrongly assigned the 1.63
signal.
In addition, the absence of a proton signal characteristic of the
terminal () trans-methyl group of the polyisoprenoid chain at
1.57 (Wolucka et al., 1996a) points to the fact that the
mycobacterial octahydroheptaprenol must be saturated at the
-end. This observation was further confirmed by positive-ion
desorption electron impact (DEI) mass spectrometry of
C35-P-mannose. In contrast to the fully unsaturated decaprenyl-P
of mycobacteria which produced, by DEI-MS, the most intense
fragment at m/z 69 (Wolucka and de Hoffmann, 1994) characteristic of unsaturated -residue, the DEI mass spectrum of
C35-P-mannose showed a predominant ion at m/z 57 and almost
equally abundant ions at m/z 69, 71, 83, and 85 (not shown), thus
indicating that the -isoprene residue of octahydroheptaprenol is
saturated (Wellburn et al., 1967).
Because of too small amounts of sample, 1H-NMR analysis of
C50-decaprenyl-P-mannose was not performed. However, the
stereochemistry of the mycobacterial decaprenol was described
previously for the C50-decaprenyl-P-arabinose derivative as ,
mono-trans, octa-cis-prenol (Wolucka et al., 1994), and the
anomeric linkage of decaprenyl-P-mannose has been determined
as (Figure 3) by our tandem mass spectrometry method
(Wolucka et al., 1996b, 1998).
Figure 5 shows the proposed formula for -D-mannopyranosyl-1-monophospho-(C35)octahydroheptaprenol of M.smegmatis.
After the isolation of 6-O-mycoloylated form of C35-P-mannose from M.smegmatis, a doubt has emerged if the free

compound, i.e., C35-P-mannose, does exist as such in the

mycobacterial cell and, in another way, if its presence in
mycobacterial preparations is not just an experimental artefact
resulting from alkaline hydrolysis step included in the purification procedure of polyprenyl-P-sugars (Besra et al., 1994). To
check this, we compared the mild-acid labile mannose content in
the DEAE-cellulose fraction of in vivo 14C-labeled mycobacterial
polyprenyl-P-sugars before and after alkaline hydrolysis
(Table I). If the DEAE-cellulose fraction of polyprenyl-P-sugars
contained 6-O-mycolate esters of C35-P-mannose, then it could
be expected that after alkaline hydrolysis much more radioactive
mannose would be released to the water phase by a mild acid. As
shown in Table I, this is not the case since almost identical
amounts of radioactive mannose were freed by mild acid from the
alkali-treated and untreated samples. The fact that mycoloylated
species are apparently absent from our polyprenyl-P-sugar
preparation is probably due to the use of more polar conditions for
the organic extraction (85% ethanol instead of a 2:1 mixture of
CHCl3-CH3OH) which could result in a selective removal of
more polar lipids and leaving behind more hydrophobic ones,
such as mycolate esters of C35-P-mannose.

PolyprenylPmannose of mycobacteria
Table II. Compositional analysis of the polyprenyl-P-sugars family of M.smegmatis
TLC band (Rf)

Compound

Yield (nmol of sugar/100 g of cells)

A (0.46)

Decaprenyl-P-arabinose

86

B (0.43)

Decaprenyl-P-ribose

340

Octahydroheptaprenyl-P-arabinose

27

C (0.39)

Decaprenyl-P-mannose

D (0.36)

Octahydroheptaprenyl-P-mannose

103

The polyprenyl-P-sugar pool was isolated from 120 g (wet weight) of M. smegmatis cells, as described in Materials and methods, and resolved by a preparative
silica TLC in CHCl3-CH3OH-NH4OH-H2O (65:25:2:2) solvent into four major bands (AD). The material eluted from each band was identified by means of
FAB-MS/MS and 1H-NMR (see text). For sugar determination, aliquots were subjected to acid methanolysis followed by trimethylsilylation and the obtained
TMS ethers of methyl glycosides were analyzed by GC-MS with perseitol, as internal standard.

Discussion

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It had been known since early seventies that mycobacteria

synthesize two distinct forms of polyprenyl-P-mannose that
contain either of the two unusual polyprenyl chains: a fully
unsaturated C50-decaprenyl or a partially saturated C35-octahydroheptaprenyl moiety (Takayama and Goldman, 1970; Takayama et al., 1973). This early evidence was based on in vitro
14C-mannosyl transfer reactions from GDP-[14C]mannose to
polyprenyl phosphates present in membrane preparations from
mycobacteria. The in vitro approach led researchers to a false
picture according to which the major form of polyprenyl-P-mannose in mycobacteria was the decaprenyl-P derivative (Takayama
et al., 1973; Yokoyama and Ballou, 1989). Moreover, structural
details about the stereochemistry of mannosyl and lipid moieties
remained unknown for a long time and the enzymes involved in
the synthesis of mannosyl-P-polyprenols have never been
purified nor characterized. Until recently, it was generally
accepted that mannosyl-P-polyprenols of mycobacteria serve as
mannosyl donors for the synthesis of cell-wall glycoconjugates
such as phosphatidylinositolmannosides, mannans/lipomannans,
and arabinomannans/lipoarabinomannans, although the only
direct experimental proof was presented by Yokoyama and
Ballou (1989) who showed the in vitro formation of free (16)
oligomannosides by using mycobacterial membrane preparations
as an enzyme source and, as a donor, a purified, enzymatically
synthesized [14C]mannosyl-P-decaprenol of plant origin.
The present work describes the isolation and structural
characterization
of
-D-mannopyranosyl-1-monophospho-(C35)octahydroheptaprenol of Mycobacterium smegmatis.
The evidence is presented that this compound is the major form
of polyprenyl-P-mannose in mycobacteria and that it exists as
such (with no mycolate attached) within the endogenous
polyprenyl-P-sugar pool. Incidentally, we demonstrated that all
three isoprene units of the mycobacterial octahydroheptaprenol
have Z (cis) configuration and that, in contrast to the claims of
Besra et al. (1994), no E (trans) isoprene residue is present within
the lipid chain.
It is noteworthy that similar to the mycobacterial octahydroheptaprenol, partially saturated at the -end dolichols were found
in fungi (Stone et al., 1967) and their phosphorylated forms also
served as acceptors of mannosyl from GDP-mannose in in vitro
assays with Aspergillus niger particulate enzyme preparations
(Barr and Hemming, 1972). The function of these compounds in
fungi is not known.
The existence in mycobacteria of two separate populations of
polyprenyl-P-mannose, C35-P-mannose and C50-P-mannose,
implies the action of highly specific mannosyltransferases able to

recognize the structural details of the lipid part. These enzymes

will probably comprise a GDP-Man : octahydroheptaprenyl-P
mannosyltransferase, a GDP-Man : decaprenyl-P mannosyltransferase and a set of specific decaprenyl-P-Man : final acceptor
mannosyltransferases. Studies with eukaryotic systems demonstrated that mannosyltransferases which employ polyisoprenyl
phosphate as a glycosyl or glycosyl-P acceptor (Rush et al.,
1997), and those utilizing dolichyl-P-mannose as a mannosyl
donor for the synthesis of glycosylphosphatidylinositols (DeLuca
et al., 1994), oligosaccharide-PP-dolichol (Rush et al., 1993), and
yeast O-mannosylated glycoproteins (Dotson et al., 1995)
discriminate between the structural variants of the polyisoprenoid
substrate that differ by the chain length and/or the presence of a
saturated -isoprene unit. The mycobacterial mannosyltransferases,
however, have not been identified.
Studies on mycobacterial polyprenyl-P-pentoses (Wolucka et
al., 1994; Wolucka and de Hoffmann, 1995) pointed to decaprenyl-P as the principal component and, thus, to a clear discrimination between the two polyprenyl phosphates in mycobacteria. An
idea has emerged (Wolucka, 1995) that the mycobacterial
mannosyl-P-polyprenols could play different biological roles.
This concept is in agreement with the identification of a
mycolyl-6-O-mannosyl-P-octahydroheptaprenol in M.smegmatis (Besra et al., 1994) which suggests the involvement of
C35-P-mannose in mycolyl transfer reactions. In addition, the fact
that, apart from C35-P-mannose, the only other polyprenylP-sugar containing a C35-octahydroheptaprenyl residue is the
-D-arabinosyl derivative (C35-P-arabinose; Wolucka and de
Hoffmann, 1995) indicates that this compound too might undergo
a mycoloylation and, then, participate in the biosynthesis of the
peptidoglycan-arabinogalactan-mycolate complex, possibly as a
donor of preformed mycoloyl-5-O-arabinofuranosyl groups of
the 5-mycoloylated, nonreducing pentaarabinofuranoside motifs
of the cell-wall arabinogalactan-mycolate complex (McNeil et
al., 1991). The reason for that such a derivative has not been
isolated so far could be inadequate conditions of the glycolipid
extraction and purification.
According to a growing body of experimental evidence, the
mannosyl-P-polyprenols of mycobacteria have apparently different functions in the cell, i.e., the minor C50-P-mannose component is a mannosyl donor in reactions catalyzed by mannosyltransferases (Yokoyama and Ballou, 1989), whereas the major
C35-P-mannose component serves as an acceptor and a donor of
mycolic acid residues (Besra et al., 1994) or, rather, as a
membrane anchor for preformed mycolic acids. Therefore, it is
conceivable that, in order to recycle C35-octahydroheptaprenyl
phosphate, mycobacteria might develop a pathway for degradation of C35-P-mannose. Such a pathway might involve a specific

B.A.Wolucka and E.de Hoffmann

960

otes (Bugg and Brandish, 1994) would not hold anymore.

Incidentally, in the case of mycobacterial polyisoprenols, the
situation is even more complex and many questions remain
unanswered. The uncommon polyprenyl-P-sugar derivatives of
mycobacteria have been fully characterized (Wolucka et al.,
1994; Wolucka and de Hoffmann, 1995; this work) and shown to
be involved in unusual reactions (Besra et al., 1994; Sherman et
al., 1996). Ironically, the isoprenoid lipid of the classical
intermediates, such as those containing a pentapeptide-muramyl
or an N-acetylglucosamine-oligosaccharide residue (Mikusova et
al., 1996), has not been characterized. Another open question is
the nature of the lipid precursor for the biosynthesis of
glycoproteins in mycobacteria (Schultz and Takayama, 1975).
Materials and methods
Bacteria
Mycobacterium smegmatis, strain 1515 from the Trudeau Mycobacterial Collection, was grown at 37
C on Nutrient Broth
(Gibco Ltd., Paisley, Scotland) with shaking for 20 h. For labeling
experiments, bacteria were grown on GAS medium (pH 6.6)
containing per liter: 0.3 g Bacto Casitone (Difco Laboratories,
Detroit, MI), 0.05 g ammonium iron (III) citrate, 4 g potassium
phosphate monobasic, 2 g citric acid, 1 g L-alanine, 1.2 g
magnesium chloride hexahydrate, 0.6 g potassium sulfate, 2 g
ammonium chloride, 1% glycerol (v/v), and supplemented with
1 mM glucose.
Isolation of C35 -octahydroheptaprenyl-P-mannose
The polyprenyl-P-sugar pool from M. smegmatis cells was
prepared as described previously (Wolucka et al., 1994), and
resolved by preparative Silica TLC in chloroform/methanol/concentrated aqueous ammonia-water (65:25:2:2). Spots were located with iodine vapors and the slowly migrating, -naphtholpositive bands (Rf 0.36 and 0.39) were scraped off, eluted with
chloroform/methanol (2:1), and reapplied to DEAE-cellulose,
which was developed with 0.05 M ammonium formate in
methanol. Salts were removed by partitioning the material in
chloroform/methanol/water (8:4:3). The organic phase was
evaporated to dryness yielding the purified C35-P-mannose (Rf
0.36) and C50-decaprenyl-P-mannose (Rf 0.39).
Preparation of

14C-labeled

polyprenyl-P-sugars

M.smegmatis cells grown on GAS medium supplemented with

glucose were centrifuged, washed, suspended in 100 ml of GAS
medium devoid of glucose and, subsequently, incubated at 37
C
for 5 min with 500 Ci of D-[14C]glucose (specific activity: 292
mCi/mmol) (Amersham Intern. plc, England). Radiolabeled cells
were harvested by centrifugation and extracted with hot 95%
ethanol at 70
C for 15 min. After further centrifugation,
supernatants (ethanolic extracts) were withdrawn, evaporated to
dryness and partitioned into a mixture of chloroform/methanol/
water (8:4:3). The organic layers were washed once with the
theoretical upper phase, evaporated to dryness, and submitted to
a small column of DEAE-cellulose. The column was washed with
5 volumes of a chloroform/methanol (2:1) solvent, and
[14C]polyprenyl-P-sugars were eluted with 3 volumes of 30 mM
ammonium formate in methanol. The eluate was desalted, a part
was saved for the analysis of mild acid-labile sugars, and the rest
was submitted to a mild alkaline hydrolysis to destroy mild
alkali-labile lipids. Alkali-stable lipids were rechromatographed
on a DEAE-cellulose column, as described above. After desalt-

Downloaded from http://glycob.oxfordjournals.org/ by guest on November 21, 2014

hydrolase able to distinguish between C50-decaprenyl- and

C35-octahydroheptaprenyl-P-mannose and to cleave the mannosyl of C35-P-mannose. We observed that mycobacterial membranes, when incubated with individual 14C-labeled C50-P-arabinose, C50-P-ribose, and C35-P-mannose, liberated to the aqueous
phase about 20% of the label from the C35-P-[14C]mannose lipid
but were inactive toward polyprenyl-P-pentoses from the same
source (only 12% of the radioactivity was found in water-soluble
products; Wolucka, unpublished observations). This fact and the
proposed role of C35-P-mannose as mycoloyl and not mannosyl
carrier (Besra et al., 1994) suggest that mycobacteria might
possess a specific, membrane-bound hydrolase responsible of the
removal of a mannosyl residue from the octahydroheptaprenylP-mannose. Specific enzymatic degradation of activated sugar
precursors has been reported. For example, specific hydrolases
are known to act on nucleotide sugar substrates such as
GDP-mannose and GDP-glucose and, thus, to participate in the
regulation of cell wall biosynthesis in E.coli (Frick et al., 1995)
and yeast (Sonnino et al., 1966), respectively. In addition, a
catabolic pathway of lipid intermediates was described in
eukaryotes (Cacan et al., 1989) and involves a novel oligosaccharide-PP-dolichol-specific pyrophosphatase (Belard et al.,
1988).
Similar substantial release to the water phase of 14C-label from
polyprenyl-P-[14C]mannose by mycobacterial membranes at the
absence of added acceptor was observed by two other groups. In
1973, Forsee and Elbein demonstrated a striking difference in the
activity of membrane preparations of mycobacteria when compared to those of plant origin, towards a set of [14C]mannosylP-polyprenols. Without addition of a water-soluble acceptor,
plant membranes were unable to transfer the 14C-label to
water-soluble products, whereas mycobacterial membranes were
able to do that. To explain these results, Forsee and Elbein argued
that, in contrast to plant membranes, those of mycobacteria are
rich in GDP, and that the latter compound can serve, therefore, as
an endogenous acceptor of a mannosyl residue from polyprenylP-mannose in a reverse reaction catalyzed by a synthase. In 1997,
by using mycobacterial membrane preparations labeled in situ
with GDP-[14C]mannose and a cell-wall fraction as a mannosyl
acceptor, Besra and colleagues reported a formation of a linear
14C-lipomannan, an (16) mannooligosaccharide glycosidically linked to phosphatidylinositol. An important loss of the
label at the absence of the acceptor was observed (Besra et al.,
1997). Unfortunately, the authors used a mixture of in vitro
formed polyprenyl-P-mannoses and did not test the nature of the
14C-mannosyl released at the absence of the cell-wall fraction.
Thus, the specific role and the metabolic fate of C35-octahydroheptaprenyl-P-mannose in mycobacteria remain unclear.
Another line of evidence for the structure/function relationships of polyisoprenyl phosphates comes from the studies on
glycoprotein-producing bacteria (Lechner et al., 1985; Hartmann
et al., 1993; Kuntz et al., 1997). In Bacillus sp., for example, two
kinds of C55-polyisoprenyl phosphates have been identified: a
fully unsaturated C55-undecaprenyl-P and an -saturated
C55-dolichyl-P (Hartmann et al., 1993). The former compound is
involved in the formation of cell-wall polysaccharides, whereas
the latter is thought to participate in the glycoprotein biosynthesis.
It is possible, therefore, that -saturated dolichyl phosphates are
common substrates for the biosynthesis of glycoproteins in both
eukaryotes and prokaryotes, whereas -unsaturated polyprenyl
phosphates are employed in the synthesis of wall polysaccharides. Consequently, the generally accepted statement that dolichols are characteristic of eukaryotes and polyprenols of prokary-

PolyprenylPmannose of mycobacteria

ing, the [14C]polyprenyl-sugars were resolved by silica TLC in

chloroform/methanol/water (65:25:4). The plate was autoradiographed and the radioactive bands comigrating with authentic,
unlabeled polyprenyl-P-sugars purified from M.smegmatis were
excised and eluted with chloroform/methanol (2:1), thus yielding
the purified [14C]-labeled decaprenyl-P-arabinose, decaprenylP-ribose, and C35-octahydroheptaprenyl-P-mannose.
Analytical procedures

Acknowledgments
We thank Professor Jean-Marie Dereppe from the Department of
Chemistry (University of Louvain) for protonNMR measurements.
Abbreviations
a.m.u., atomic mass unit; C50-P, decaprenyl phosphate; C50-PMan, decaprenyl-P-mannose; C35-P, octahydroheptaprenyl phosphate; C35-P-Man, octahydroheptaprenyl-P-mannose; TLC, thin
layer chromatography; GC-MS, gas chromatographymass spec-

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Methanolysis and trimethylsilylation of intact glycolipids was

performed as described earlier (Wolucka et al., 1993). Briefly, a
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which was then introduced into the ion source and heated close
to the electron beam.

trometry; FAB-MS/MS, fast atom bombardmenttandem mass

spectrometry.

B.A.Wolucka and E.de Hoffmann

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