Physicochemicalcharacterizationandbiological activity
of aglycoglycerolipid from
Mycoplasma fermentans
Klaus Brandenburg
1
, Frauke Wagner
1
, Mareike Mu¨ ller
1
, Holger Heine
1
,Jo¨ rg Andra¨
1
, Michel H. J. Koch
2
,
Ulrich Za¨ hringer
1
and Ulrich Seydel
1
1
Forschungszentrum Borstel, Center for Medicine and Biosciences, Borstel;
2
European Molecular Biology Laboratory,
Outstation Hamburg, Hamburg, Germany
We report a comprehensive physicochemical characteriza-
tion ofaglycoglycerolipidfromMycoplasma fermentans,
MfGl-II, in relation to its bioactivity and compared this with
the respective behaviors of phosphatidylcholine (PC) and a
bacterial glycolipid, lipopolysaccharide (LPS) from deep
rough mutant Salmonella minnesota strain R595. The b«a
gel-to-liquid crystalline phase transition behavior of the
hydrocarbon chains with T
c
¼ 30 °C for MfGl-II as well as
for LPS exhibits high similarity between the two glycolipids.
A lipopolysaccharide-binding protein (LBP)-mediated
incorporation into negatively charged liposomes is observed
for both glycolipids. The determination of the supramole-
cular aggregate structure confirms the existence ofa mixed
unilamellar/cubic structure for MfGl-II, similar to that
observed for the lipid A moiety of LPS. The biological data
clearly show that MfGl-II is able to induce cytokines such as
tumor necrosis factor-a (TNF-a) in human mononuclear
cells, although to a significantly lower degree than LPS. In
contrast, in the Limulus amebocyte lysate test, MfGl-II is
completely inactive, and in the CHO reporter cell line it does
not indicate any reactivity with the Toll-like receptors TLR-
2 and -4, in contrast to control lipopeptides and LPS. These
data confirm the applicability of our conformational concept
of endotoxicity to nonlipid A structures: an amphiphilic
molecule with a nonlamellar cubic aggregate structure cor-
responding to a conical conformation of the single molecules
and a sufficiently high negative charge density in the back-
bone.
Keywords: glycolipid; lipopolysaccharide; endotoxic con-
formation; cytokine induction; Limulus amebocyte lysate
(LAL) assay.
Mycoplasma fermentans is a member of the class Mollicutes,
which comprises wall-less procaryotes. Mycoplasmas are
pathogens infecting a broad spectrum of diverse hosts such
as animals, plants and humans, where they cause several
invasive or chronic diseases [1–3]. M. fermentans was first
isolated from the human urogenital tract [4], and since then
its role as pathogen and cofactor in diverse diseases has
emerged, in particular its role in the pathogenesis of
rheumatoid arthritis [5]. In recent years it was suggested
that M. fermentans is involved in triggering the develop-
ment of AIDS in HIV-positive individuals, acting as a
cofactor in pathogenesis [6]. Although little is known about
the molecular mechanisms underlying M. fermentans
pathogenicity, it is reasonable to assume that the inter-
actions with host cells are mediated by components of its
plasma membrane [7–9]. Matsuda et al.isolatedtwo
phosphocholine-containing glycoglycerolipids [10] and
elucidated the structure of one as 6¢-O-phosphocholine-
a-glucopyranosyl-(1¢-3)-1,2-diacyl-sn-glycerol (MfGl-I) [11].
Recently, we identified and characterized a major glyco-
glycerolipid from the membrane of M. fermentans which
was found to be 6¢-O-(3¢¢-phosphocholine-2¢¢-amino-
1¢¢-phospho-1¢¢,3¢¢-propanediol)-a-
D
-glucopyranosyl-(1¢-3)-
1,2-diacyl-sn-glycerol (MfGl-II) [12]. Furthermore, we
could show that MfGl-II triggers inflammatory response
in primary rat astrocytes such as activation of protein kinase
C, secretion of nitric oxide and prostaglandin E2 as well as
augmented glucose utilization and lactate formation [11].
These data were supported by others [13,14].
From these findings, the elucidation of molecular
mechanisms underlying or mediating these activities on a
molecular level should be of high interest. It has been
reported for other glycolipids from the outer membrane,
in particular for bacterial lipopolysaccharides (LPS), that
their biologicalactivity is connected with a particular
physicochemical behavior of these molecules, which relates
to their molecular shape, the intra- and intermolecular
conformation, and their property to be transported by
lipid transfer proteins such as lipopolysaccharide-binding
protein (LBP) [15–17]. Therefore, we wanted to know if
similar characteristics hold also for MfGl-II, i.e. whether
there is a general principle connecting physicochemical
parameters andbiologicalactivityof glycolipids to
different structures.
Correspondence to K. Brandenburg, Forschungszentrum Borstel,
Division of Biophysics, D-23845 Borstel, Germany.
Fax: +49 4537 188632, Tel.: +49 4537 188235,
E-mail: kbranden@fz-borstel.de
Abbreviations: FTIR, Fourier transform infrared; FRET, fluorescence
resonance energy transfer; H, hexagonal; LAL, Limulus amebocyte
lysate; LBP, lipopolysaccharide-binding protein; LPS, lipopolysac-
charide; MALP, macrophage-activating lipopeptide; MfGl-I,
6¢-O-phosphocholine-a-glucopyranosyl-(1¢,3)-1,2-diacyl-sn-glycerol;
MNC, mononuclear cell; PC, phosphatidylcholine; PS,
phosphatidylserine; TNF-a, tumor necrosis factor a.
(Received 2 April 2003, accepted 13 June 2003)
Eur. J. Biochem. 270, 3271–3279 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03719.x
Based on the well characterized primary structure,
the present paper describes the physicochemical proper-
ties of MfGl-II and its ability to induce cytokines in
human mononuclear cells. In the present paper, Fourier
transform infrared (FTIR) spectroscopy was applied to
determine the phase behavior via the analysis of the
peak position of the symmetric stretching vibration of
the methylene groups. Additionally, this technique was
applied to monitor the conformation of headgroup
moieties such as phosphate. The data obtained for
MfGl-II are related to those from LPS and phospha-
tidylcholine (PC) and show characteristic differences
between these amphiphiles. Synchrotron radiation small-
angle X-ray diffraction was applied for the determination
of the aggregate structure, andfrom the diffraction
patterns the existence of mixed unilamellar/nonlamellar
aggregate structures are deduced similar to those observed
for lipid A. We furthermore show by fluorescence reso-
nance energy transfer (FRET) technique that, analogously
to LPS, intercalation of MfGl-II in negatively charged
membrane systems such as liposomes made from phos-
phatidylserine (PS) can be mediated by lipopolysaccha-
ride-binding protein (LBP). In biological tests, we can
show that MfGl-II is able to induce tumor necrosis factor-
a (TNF-a) in human mononuclear cells, whereas in the
LPS-specific Limulus amebocyte lysate test no activity is
observed, which indicates that no LPS contamination is
present.
With these data, our conformational concept of endo-
toxicity [17] can for the first time be successfully applied also
to a nonlipid A structure.
Materials and methods
Growth of bacteria
Mycoplasma fermentans strain PG18 was grown in a
modified Channock medium inoculated with 2% of a
48-h culture and incubated statically at 37 °C as described
previously [12]. After 68 h, cells were harvested, washed
twice with 0.25
M
NaCl in 10 m
M
Tris/HCl, pH 7.4, and
freeze dried as described previously [12] with yields
ranging from 160 to 200 mg dry weight per liter of
medium.
Lipid extraction and purification
Freeze-dried cells were suspended in 25 m
M
Tris/HCl
buffer, pH 7.5, containing 0.25
M
NaCl to obtain a final
concentration of 25 mg cellsÆmL
)1
. Lipids were extracted
from the cell suspension by the method of Bligh and Dyer
[18], and the organic layer was concentrated to dryness on a
rotary evaporator. Lipids (0.2 gÆg
)1
dried cells) were redis-
solved in chloroform/methanol 1 : 4 (v/v) to a concentra-
tion of 40 mgÆmL
)1
. Quantitative separation of polar and
nonpolar lipids was achieved by HPLC on Nucleosil
column (10 · 500 mm, Nucleosil 50-7, Macherey-Nagel).
Crude lipid extracts (20 mg) were applied to the column and
eluted with a linear gradient of solvent A (chloroform/
methanol 1 : 4, v/v) and solvent B (chloroform/methanol/
water 1 : 4 : 2.5, v/v/v) starting with 0% solvent B for
30 min, then stepwise increasing to 15% B (150 min), 50%
B (10 min), holding 20 min 50% solvent B at a flow rate of
2mLÆmin
)1
(35 bar). Fractions were collected for 2 min
each and analyzed by TLC (chloroform/methanol/water
100 : 100 : 30, v/v/v). MfGl-II eluted as the last lipid,
appropriate fractions (nos 36–60) were combined,
R
f
¼ 0.17 (yield 4.16 mg).
Lipid samples
LPS from deep rough mutant Re from Salmonella
minnesota strain R595 was extracted according to PCP I:
2% phenol/5% chloroform/8%petrol ether, v/v) proce-
dure [19], purified by treatment with DNAse/RNAse and
proteinase K, and lyophilized and used in the natural salt
form. The lipopeptide palmitoyl-3-cysteine-serine-lysine-4
(Pam3CSK4) and the macrophage-activating lipopeptide-2
(MALP-2) were kind gifts of K H. Wiesmu
¨
ller
1
(Tu
¨
bingen,
Germany). Bovine brain 3-sn-PS and egg 3-sn-PC were
obtained from Sigma (Deisenhofen, Germany). For pre-
paration of liposomes froma phospholipid mixture
corresponding to the composition of the macrophage
membrane (PLMN), PS or PC the lipids were solubilized
in chloroform, the solvent was evaporated under a stream
of nitrogen, and the lipids were resuspended in the
appropriate volume of NaCl/P
i
and further treated as
described for LPS.
Glycolipid preparations
The MfGl-II and LPS samples were prepared by directly
suspending an appropriate amount of lipid into buffer,
vortexing for some minutes, heating to 60 °C, again
vortexing, and recooling to 10 °C. This procedure was
repeated twice.
b«a gel to liquid crystalline phase transition
Fourier-transform infrared (FT-IR) spectroscopic measure-
ments were performed on a Bruker IFS-55 (Bruker Instru-
ments, Karlsruhe Germany) with a 10
)2
M
lipid suspension
prepared as described above. The phase behavior of the acyl
chains was derived from the peak position of the symmetric
stretching vibration of the methylene groups m
s
(CH
2
),
which lies around 2850 cm
)1
in the gel and between
2852 cm
)1
and 2853 cm
)1
in the liquid-crystalline phase
[20,21].
Lipid headgroup conformation
For acharacterizationof the conformation of functional
groups within the lipid backbones such as the phosphate,
lipid suspensions were prepared as described above.
Subsequently, 10 mL were spread on a CaF
2
crystal and
allowed to stand at room temperature until all free water
was evaporated. After this, IR spectra were recorded at
room temperature and at 37 °C. Usually, the original
spectra were evaluated directly anda spectral analysis was
performed in the fingerprint region between 1800 and
900 cm
)1
. In the case of overlapping absorption bands,
either resolution enhancement techniques like Fourier self-
deconvolution [22] or a curve-fit analysis [23] were
performed.
3272 K. Brandenburg et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Aggregate structures and molecular shape
For the determination of the three-dimensional supra-
molecular structure of the lipid aggregates, X-ray diffraction
measurements were performed at the European Molecular
Biology Laboratory outstation at the Deutsches Elektronen
Synchrotron (DESY) in Hamburg as described [24] using
the double focusing monochromator-mirror camera X33
[25]. In the diffraction patterns, the logarithm of the
diffraction intensity log I is plotted against the scattering
vector s (s ¼ 2sinh/nk ;2h, scattering angle; k ¼ 0.15 nm,
wavelength), and the X-ray spectra were evaluated as
described previously [21]. From the spacing ratios of the
diffraction maxima an assignment to defined three-dimen-
sional aggregate structures is possible, i.e. to lamellar,
nonlamellar cubic, and inverted hexagonal II (HII). From
this, the conformation of the individual molecules can be
approximated [17,24], which is cylindrical in the case of
lamellar structures, the cross-sections of the hydrophilic and
hydrophobic moieties are identical, and conical or wedge-
shaped in the case of nonlamellar cubic and direct HI or
inverted HII structures; the cross-sections of the single
portions are different.
LBP-mediated intercalation of lipids into phospholipid
membranes
FRET was performed as described earlier [26]. Briefly,
phospholipid liposomes corresponding to the composition
of the macrophage membrane or from pure PC or PS
were double-labeled with the fluorescent dyes N-(7-nitro-
benz-2-oxa-1,3-diazol-4-yl)-phosphatidylethanolamine
(NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phos-
phatidylethanolamine (Rh-PE) (Molecular Probes, Eugene,
OR, USA), respectively. Intercalation of unlabeled mole-
cules into the double-labeled liposomes leads to probe
dilution and with that to a decrease in the efficiency of
FRET: the emission intensity of the donor increases
and that of the acceptor decreases (for clarity, only the
donor emission intensity is shown). The double-labeled
liposomes were preincubated with unlabeled LPS and
recombinant human lipopolysaccharide-binding protein
LBP was added.
Endotoxin activity determination by the chromogenic
Limulus test
Endotoxin activityof the glycolipids was determined by a
quantitative kinetic assay [27] based on the reactivity of
Gram-negative endotoxin with Limulus amebocyte lysate
(LAL) using test kits from BioWhittaker (# 50–650 U).
Induction of tumor necrosis factor-a
For the isolation of mononuclear cells (MNC), blood was
taken from healthy donors and heparinized (20 IEÆmL
)1
).
The heparinized blood was mixed with an equal volume of
Hank’s balanced salt solution and centrifuged on a Ficoll
density gradient for 40 min (21 °C, 500 g)
2
.Theinterphase
layer of mononuclear cells was collected and washed
three times in serum-free RPMI 1640 containing 2 m
M
L
-glutamine, 100 UÆmL
)1
penicillin, and 100 mgÆmL
)1
streptomycin. The cells were resuspended in serum-free
medium, and their number was adjusted to 5 · 10
6
mL
)1
.
For stimulation, 200 mL per well heparinized MNC
(5 · 10
6
mL
)1
) were filled into 96-well culture plates and
stimulated with endotoxins in serum-free medium. The
stimuli were serially diluted in serum-free RPMI 1640 and
added to the cultures at 20 mL per well. The cultures were
incubated for 4 h at 37 °C and 5% CO
2
. Supernatants were
collected after centrifugation of the culture plates for 10 min
at 400 g andstoredat)20 °C until determination of
cytokine concentration.
The immunological determination of TNF-a in the
cell supernatants was determined in a sandwich-ELISA.
Ninety-six-well plates (Greiner, Solingen, Germany) were
coated with a monoclonal antibody against TNF (clone
6b from Intex, Germany). Cell culture supernatants and
the standard (recombinant TNF, Intex) were diluted with
buffer. The plates were shaken 16–24 h at 4 °C.Forthe
removal of free antibodies, the plates were washed six
times in distilled water. Subsequently, the color reaction
was started by addition of tetramethylbenzidine in alco-
holic solution and stopped after 5–15 min by addition of
0.5
M
sulfuric acid. In the color reaction, the substrate is
cleaved enzymatically, and the product can be measured
photometrically. This was carried out on an ELISA
reader (Rainbow, Tecan, Crailsham, Germany) at a
wavelength of 450 nm, and the values were related to
the standard.
Cell lines
The CHO/CD14 reporter line, clone 3E10, is a stably
transfected CD14-positive CHO cell line that expresses
inducible membrane CD25 (Tac antigen) under transcrip-
tional control of the human E-selectin promoter (pE-
LAM.Tac [28]). The CHO/CD14/huTLR2 (3E10TLR2)
reporter cell line was constructed by stable cotransfection of
3E10 with the cDNA for human TLR2 and pcDNA3
(Invitrogen), as described [29]. CHO cell lines were grown in
Ham’s F12 medium containing 10% fetal bovine serum and
1% penicillin/streptomycin at 37 °C in a humidified 5%
CO
2
environment. Medium was supplemented with
400 UÆmL
)1
hygromycin B and 0.5 mgÆmL
)1
G418
(3E10TLR2).
Flow cytometry analysis of NF-6B activity
CHO reporter cells were plated at a density of 2.5 · 10
5
per
well in 24-well dishes. The following day, the cells were
stimulated as indicated in Ham’s F12 medium containing
10% fetal bovine serum (total volume of 0.3 mL per well).
Subsequently, the cells were harvested with trypsin-EDTA,
labeled with FITC-CD25 mAb (Dako, Germany) and
analyzed by flow cytometry, as previously described [28].
Results
The chemical structures for MfGl-II, lecithin (PC), and LPS
from S.minnesotaR595 are shown in Fig. 1A,B, respect-
ively. MfGl-II and PC both have a diacyl hydrophobic
moiety and an identical phosphocholine headgroup. Like
LPS, MfGl-II carries two negatively charged phosphates
Ó FEBS 2003 Biophysical study ofMycoplasma glycolipid (Eur. J. Biochem. 270) 3273
and a saccharide moiety which, however, is differently
linked to the acyl chains in the two molecules.
Gel-to-liquid crystalline phase transition and lipid
headgroup conformations
The determination of the b«a gel-to-liquid crystalline
phase transition of the acyl chains from the evaluation of the
symmetric stretching vibration of the methylene groups m
s
(CH
2
) revealed a phase transition at around 30 °C for
MfGl-II similar to that of deep rough mutant LPS from
S. minnesota strain R595 (Fig. 2). The entire phase behavior
of MfGl-II and LPS is very similar except that the
wavenumber values are lower for the latter indicating a
slightly higher overall acyl chain order. In contrast, natural
PC exhibits high wavenumber values over the entire
temperature range, from which the existence of only the
unordered a-phase can be concluded.
The infrared spectrum of MfGl-II in the fingerprint
region (Fig. 3a) displays strong bands at 1739 cm
)1
corres-
ponding to the ester double bond stretching m (C ¼ O), the
lipid scissoring band d (CH
2
) at 1465 cm
)1
,theantisym-
metric and symmetric stretching vibrations of the negatively
charged phosphate groups m
as
(PO
2
–
)at1220cm
)1
and m
s
(PO
2
–
) at 1120 cm
)1
, respectively [23,30], and the bands at
1172, 1085, and 1038 cm
)1
assigned to glucose ring
vibrations [31]. As no additional bands in the range of the
amide vibrations centered at 1650 and 1550 cm
)1
can be
observed, any significant contamination by proteins or
Fig. 1. Chemical structures of PC, glycolipid from M. fermentans
MfGl-II, and LPS Re from S. minnesota strain R595.
Fig. 2. Peak position of the methylene stretching vibration m
s
(CH
2
)in
dependence on temperature illustrating the b«a gel-to-liquid crystalline
phase transition for phosphatidylcholine, MfGl-II, and LPS Re.
Fig. 3. Infrared spectrum in the spectral range 1800–900 cm
)1
(A) and
in the range of the negatively charged phosphate band m
as
(PO
2
–
) 1300–
1190 cm
)1
(B)ofhydratedPC,MfGl-II,andLPSRe.
3274 K. Brandenburg et al. (Eur. J. Biochem. 270) Ó FEBS 2003
lipopolysaccharides can be excluded. The band contour of
m
as
(PO
2
–
) was analyzed after baseline subtraction (Fig. 3b)
and revealed strong differences between MfGl-II, LPS Re,
and PC (in this case dimyristoyl PC).
LPS exhibits two band components, one at higher
wavenumber (1260 cm
)1
) with relatively low bandwidth,
corresponding to phosphate with low hydration, and one
broader band component (at 1223 cm
)1
), corresponding to
higher hydration [23]. The spectrum for PC shows the
occurrence of one major band around 1225 cm
)1
in
accordance with the well-known high water-binding capa-
city of lecithin headgroups [30]. Finally, MfGl-II exhibits a
main band at 1245 cm
)1
and further weak bands at 1220
and 1260 cm
)1
. Thus, the phosphate groups of this glyco-
lipid are more strongly hydrated than LPS but less than PC.
LBP-mediated intercalation into target cell membranes
The intercalation of MfGl-II and LPS into phospholipid
liposomes by the transport protein LBP was investigated by
FRET spectroscopy. Figure 4 illustrates that there is an
increase of the NBD-fluorescence intensity immediately
after the addition of LBP to preincubated PS in the presence
of MfGl-II or LPS (Fig. 4A) which indicates a swelling of
the PS-liposomes due to the incorporation of the glycolipids.
For the PLMN system, also an incorporation of both
glycolipids takes place, but to a lesser degree than for the
PS-liposomes (data not shown).
In contrast, only a very small intensity increase due to
incorporated LPS, but even a decrease in fluorescence
intensity corresponding to a dilution, i.e. no incorporation
of MfGl-II into pure zwitterionic PC liposomes take place
(Fig. 4B).
Supramolecular aggregate structure
For the elucidation of the three-dimensional aggregate
structure of MfGl-II synchrotron radiation X-ray small-
angle diffraction was used. The diffraction pattern in Fig. 5,
which was slightly deconvoluted to reduce noise, shows a
broad diffraction band superimposed by four weak diffrac-
tion peaks. The shape of the main reflection band located
between 0.1 and 0.3/nm can be interpreted by the existence
of a unilamellar structure. The location of the four small
peaks superimposed fit the relations 17.0 ¼ 8.48 Ö2,
16.9 ¼ 6.90 Ö6, 17.0 ¼ 4.90 Ö12, 17.0 ¼ 3.47 Ö24, which
can be assigned to a cubic structure with a periodicity
a
Q
¼ (16.95 ± 0.10) nm. The space group, however, can-
not be determined due to a lack of observable reflections.
From these findings, a superposition ofa main unilamellar
with a nonlamellar cubic structure can be deduced, which
would correspond to a very slight conical conformation of
the individual molecules with different cross-sections of the
hydrophobic and the hydrophilic moieties. From these data,
however, no unequivocal statement is possible which of
the moieties has a higher cross-section.
Fig. 4. NBD-donor fluorescence intensity as function of time of double-
labeled liposomes made from PS (A) or from PC (B) after the addition of
MfGl-IIorLPSReatt = 50 s and subsequent addition of LBP
(0.2 m
M
)att = 100 s in comparison to control NaCl/P
i
(phosphate
buffered saline). The concentration of the glycolipids, PC and PS was
10 m
M
each.
Fig. 5. Synchrotron radiation X-ray small-angle diffraction pattern of
MfGl-II at 40 °C and 85% water content. The diffraction pattern
indicates the existence ofa unilamellar structure (broad band) super-
imposed by a cubic (four small reflections) structure. The diffraction
pattern was resolution-enhanced by applying Fourier self-deconvolu-
tion ([22]; parameters: bandwidth 0.05, enhancement factor 1.5 and
Gaussian to Lorentz ratio 0.6). The cubic periodicity at 17 nm (in
parenthesis) is not directly observable, but can be calculated from the
locations of the four reflections (see text).
Ó FEBS 2003 Biophysical study ofMycoplasma glycolipid (Eur. J. Biochem. 270) 3275
LAL assay
The LAL test is based on the property of lysates of
amebocytes of the horseshoe crab Limulus polyphemus,to
form a solid gel in the presence of minute amounts of
endotoxins. The comparison of LPS and MfGl-II in the
LAL assay shows, as expected, activityof LPS in the range
of down to 10 pgÆmL
)1
, whereas MfGl-II has only activity
in the range ‡10 mgÆmL
)1
. The latter result also excludes a
significant contamination of the MfGl-II preparation by
LPS.
TNF-a induction in human mononuclear cells
As one major cytokine, which is induced by stimulating
agents in human mononuclear cells (MNCs), TNF-a was
monitored in an ELISA. In Fig. 6, the capacities of LPS and
MfGl-II to induce TNF-a in MNCs are compared. LPS
causes strong TNF-a production down to concentrations of
<1 ngÆmL
)1
, while for MfGl-II a significant response is
found down to 100 ngÆmL
)1
, thus indicating that MfGl-II,
although two orders of magnitude less active than LPS, still
induces cytokines to a significant degree.
CHO reporter system
In order to investigate the potential involvement of TLR2
and TLR4 in the recognition and signal transduction of the
glycolipids, we analyzed the stimulatory activityof MfGl-II
in a CHO cell reporter system. Upon the induction of
nuclear factor-kappa B translocation in these reporter cells,
human CD25 is expressed on the cell surface [28]. The data
(Fig. 7) clearly indicate that neither the expression of CD14
and TLR4 (3E10) nor the expression of CD14, TLR2, and
TLR4 (3E10 TLR2) is sufficient to enable the cells to
respond to MfGl-II even at the highest concentration
10 mgÆmL
)1
. As controls, stimulation of the different cell
lines with either LPS from Salmonella friedenau or the
lipopeptides from synthetic (Pam3CysSerLys4) or natural
origin (MALP-2) showed the expected phenotype, i.e. LPS
reacts essentially to TLR4, while the lipopeptide or protein
exhibit TLR2-reactivity. Thus, a possible contamination of
the MfGl-II with a lipoprotein or MALP-2 [32], which
could explain the cytokine-inducing capacity, can be
excluded.
Discussion
Mycoplasma fermentans has been reported to accompany
several diseases such as rheumatoid arthritis and HIV [6].
For the former, the mycoplasma organisms may be a
cofactor in the pathogenesis, but its precise role remains
obscure. Candidates for structures mediating pathogenicity
are molecules in the cell membrane of Mollicutes [7–9].
These are glycolipids, lipopeptides (macrophage-activating
lipopeptides, MALP), or lipoproteins. The glycoconjugates
MALP-I and -II are known to activate macrophages [33] on
a TLR-2 and MyD88- dependent pathway [34] and are
active down to picomolar concentration [32]. Also, they are
strong inducers of cytokines and chemokines.
Using anti-(MfGl-II) sera, we could show that the
terminal phosphocholine residue of MfGl-II is responsible
for the attachment of M. fermentans to host cells. The anti-
(MfGl-II) sera inhibit the attachment of M. fermentans to
Molt-3 lymphocytes suggesting that MfGl-II plays a major
role in M. fermentans–host cell interaction. As tested in an
ELISA assay, phosphocholine almost completely abolished
antibody interaction with MfGl-II suggesting that the anti-
(MfGl-II) repertoire is composed primarily of anti-phos-
phocholine Ig [8].
Here, the glycolipid MfGl-II was considered to be a likely
candidate for triggering proinflammatory reactions in
human monocytes. MfGl-II represents a species-specific
immunodeterminant of M. fermentans, as anti-(MfGl-II)
sera do not cross-react with lipid extracts of other Myco-
plasma species like Mycoplasia penetrans [8].
The comprehensive characterizationof MfGl-II from
pathogenic M. fermentans presented here yields many
surprising physicochemical similarities to the characteristics
of LPS [35]. This refers to the phase transition behavior and
the fluidity of the glycolipid chains at 37 °C (Fig. 1), the
Fig. 6. Induction of TNF-a in human mononuclear cells by MfGl-II and
LPS Re as function of glycolipid concentration. The error bar (standard
deviation) results from the determination of TNF-a in duplicate at two
different dilutions. The data are representative of three independent
measurements.
Fig. 7. Relative activation of CHO reporter cells stimulated with MfGl-
II, the lipopeptide Pam3CysSerLys4, LPS S-form from Salmonella
friedenau, the MALP-2 (macrophage activating lipoprotein), and inter-
leukin-1. The IL-1 induced expression of NF-6B reporter signal was set
to 100%.
3276 K. Brandenburg et al. (Eur. J. Biochem. 270) Ó FEBS 2003
strong LBP-induced incorporation into negatively charged
phospholipid liposomes (Fig. 4), anda diffraction pattern,
which is consistent with the existence ofa unilamellar
superimposed by a cubic structure (Fig. 5). Thus, according
to our concept of an endotoxic activity, which requires a
cubic supramolecular aggregate structure corresponding to
a conical conformation of the individual molecules and an
LBP-driven incorporation into target cell membranes, for
which a sufficiently high negative charge is needed, MfGl-II
is a candidate as immunostimulating agent. Actually, MfGl-
II induced TNF-production, although to a lower degree
than LPS (Fig. 6).
Although we presently cannot answer the question
whether the cubic structure observed is of the ÔnormalÕ
(right side out) or the inverted type, the geometry of the
molecule with its bulky headgroup is in favor of the former
type. With respect to a correlation to bioactivity, we have
observed that enterobacterial hexaacylated lipid A as well as
a triacylated lipid A derived from the former adopt an
inverted cubic or a direct micellar HI phase, respectively
[23,24,36]. Both preparations have been shown to induce
cytokines in mononuclear cells, which was not the case for
tetra- or penta-acylated lipid A with their preference for
multilamellar structures [17,35].
It is important to note that MfGl-II shows practically no
LAL activity, which excludes an LPS contamination. Such
LPS contamination can never be excluded, as in the
extraction and purification process it is very difficult to be
completely pyrogen-free (bacteria are ubiquitous).
Furthermore, the absence of amide vibrations in the
infrared spectrum of MfGl-II in the wavenumber range
1500–1700 cm
)1
(Fig. 3A) rules out a putative contamin-
ation of the MALP lipopeptide, which also is not very likely
in the light of the high negative charge density of the
headgroup of the latter, leading to different migration in the
HPLC purification process. Additionally, the absence of a
lipoprotein is confirmed by the absence of TLR2 reactivity
in the CHO reporter system (Fig. 7), which has been shown
to be responsible for signaling in the case of lipopeptides and
lipoproteins [37].
Although the TNF-inducing capacity of MfGl-II is lower
than that of LPS, it is still higher with respect to the
cytokine-inducing capacity of other bacterial activators like
glycosphingolipid from Sphingomonas paucimobilis (GSL-4,
a tetrasaccharide glycolipid with a sphingolipid anchor),
which shows activation in the range ‡1mgÆmL
)1
[38]. This
glycolipid was found to stimulate human MNCs in a CD14-
independent way, and the response could not be blocked by
antagonistic lipid A part structures, therefore indicating a
completely different activation pathway [39].
In contrast, the results from the FRET measurements
(Fig. 4) indicate a signaling pathway identical to that of
LPS. This may be explained by the fact that MfGl-II as
well as lipid A, the endotoxic principle of LPS, exhibits a
high negative charge density due to the presence of two
phosphates. GSL molecules have only one negative
charge, a glucuronic acid. Whether the kind of charge,
phosphate or uronic acid, plays a role in endotoxin
signaling, cannot be answered unequivocally. We found
earlier that a lipid A analogue in which the 1-phosphate is
substituted by a carboxymethyl group (CM-506), exhibits
the same activity as natural Escherichia coli-type lipid A
or its synthetic analogue 506 [15]. In contrast, the lipid A
from Rhodospirillum fulvum, in which the 1-phosphate is
substituted by a heptose and the 4¢-phosphate by a
galacturonic acid, is biologically, i.e. agonistically as well
as antagonistically, completely inactive. The lack of
antagonistic activity may be explained by the fact that
this lipid A does not intercalate into target cell membranes
by LBP-mediated transport [35].
We have shown recently that endotoxin aggregates are
the active units, i.e. they are at least one order of magnitude
more active than monomers [40]. Furthermore, we have
found LBP to exist in a membrane-bound form in which it is
able to cause an intercalation of LPS into this membrane
[41]. It can be assumed that membrane proteins such as
CD14 may also cause a membrane intercalation of LPS.
In the membrane, the glycolipids are expected to form
domains, because their chemical structures are completely
different from those of the phospholipids. These domains
may be formed around membrane proteins or migrate to
these after their formation, as an attractive force could be
exerted due to the high charge density and existence of polar
functional groups. At the site of the signaling protein, which
may be the Toll-like receptors (TLR2 or TLR4 [42,43])
and a potassium channel [44], only conically shaped glyco-
lipids such as lipid Aand MfGl-II represent a mechanical
disturbance leading to a conformational change of the
protein and, with that, signal transduction.
Recently, Ben-Menachem
3
et al. [45] presented a physico-
chemical characterizationof MfGl-II to study the permeab-
ility of M. fermentans. They observed also the existence of a
gel-to-liquid crystalline phase transition, which they estima-
tedtorangebetween35and45°C.Furthermore,from
31
P-NMR they proposed only lamellar phases as aggregate
structure, which was deduced from the ÔisotropicÕ signal in
the NMR experiment. This is in complete accordance to our
data indicating the existence of unilamellar vesicles as well as
a nonlamellar cubic structure, as also the latter leads to an
isotropic signal [46]. Therefore, a differentiation between
unilamellar and cubic structures is not possible using the
NMR technique.
Acknowledgments
We are indepted to G. von Busse, S. Groth, and U. Diemer for
performing the IR spectroscopic, TNF-induction and LAL activity
measurements, respectively.
This work was financially supported by the Deutsche Forschungsg-
emeinschaft (SFB 367 project B8) and by the German-Israeli
foundation for Scientific Research and Development (GIF grant
I-373-169-09/94).
References
1. Lee, I M. & Davis, R.E. (1992) Mycoplasmas with infect plants
and insects. In Mycoplasmas ) Molecular Biology and Pathogen-
esis (Maniloff, J., McElhaney, R.N., Finelli, M.R. & Baseman,
J.B., eds), pp. 379–390. American Society for Microbiology,
Washington, DC.
2. Simecka, J.W., Davis, J.K., Davidson, M.K., Ross, S.E., Sta-
dtla
¨
nder, C.T.K.H. & Cassell, G.H. (1992) Mycoplasma diseases
an animals. In Mycoplasmas – Molecular Biology and Pathogenesis
(Maniloff, J., McElhaney, R.N., Finch, L.R. & Baseman, J.B.,
Ó FEBS 2003 Biophysical study ofMycoplasma glycolipid (Eur. J. Biochem. 270) 3277
eds), pp. 391–415. American Society for Microbiology, Wash-
ington, DC.
3. Krause, D.C. & Taylor-Robinson, D. (1992) Mycoplasmas which
infect humans. In Mycoplasmas – Molecular Biology and Patho-
genesis (Maniloff,J.,McElhaney,R.N.,Finch,L.R.&Baseman,
J.B., eds), pp. 417–444. American Society for Microbiology,
Washington, DC.
4. Ruiter, M. & Wentholt, H.M.M. (1952) The occurrence of a
pleuropneumonia-like organism in fuso-spillary infections of the
human genital mucosa. J. Invest. Dermatol. 18, 313–323.
5. Williams, M.H. & Bruckner, F.E. (1971) Immunological reactivity
to Mycoplasmafermentans in patients with rheumatoid arthritis.
Ann. Rheum. Dis 30, 271–273.
6. Lo, S C. (1992) Mycoplasmas and AIDS. In Mycoplasmas –
Molecular Biology and Pathogenesis (Maniloff, J., McElhaney,
R.N.,Finch,L.R.&Baseman,J.B.,eds),pp.525–545.American
Society for Microbiology, Washington, D.C.
7. Salman, M., Deutsch, J., Tarshis, M., Naot, Y. & Rottem, S.
(1994) Membrane lipids ofMycoplasma fermentans. FEMS
Microbiol. Lett. 123, 255–260.
8. Ben-Menachem., G., Wagner, F., Za
¨
hringer, U., Rietschel, E.Th
& Rottem, S. (1997) Antibody response to MfGl-II, a phos-
phocholine-containing major lipid ofMycoplasma fermentans
membranes. FEMS Microbiol. Lett. 154, 363–369.
9. Ben-Menachem., G., Rottem, S., Tarshis, M., Barash, V. &
Brenner, T. (1998) Mycoplasmafermentans glycolipid triggers
inflammatory response in rat astrocytes. Brain Res. 803, 34–38.
10. Matsuda, K., Harasawa, R., Li, J L., Kasama, T., Taki, T.,
Handa, S. & Yamamoto, N. (1995) Identification of phos-
phocholine-containing glycoglycerolipids purified from Myco-
plasma fermentans-infected human helper T-cell culture as
components of M. fermentans. Microbiol. Immunol. 39, 307–313.
11. Matsuda, K., Kasama, T., Ishizuka, I., Handa, S., Yamamoto, N.
& Taki, T. (1994) Structure ofa novel phosphocholine-containing
glycoglycerolipid fromMycoplasma fermentans. J. Biol. Chem.
269, 33123–33128.
12. Za
¨
hringer, U., Wagner, F., Rietschel, E.Th, Ben-Menachem., G.,
Deutsch, J. & Rottem, S. (1997) Primary structure ofa new
phosphocholine-containing glycoglycerolipidofMycoplasma fer-
mentans. J. Biol. Chem. 272, 26262–26270.
13. Matsuda, K., Li, J L., Harasawa, R. & Yamamoto, N. (1997)
Phosphocholine-containing glycoglycerolipids (GGPL-1 and
GGPL-III) are species-specific major immunodeterminants of
Mycoplasma fermentans. Biochem. Biophys. Res. Commun. 233,
644–649.
14.Li,J L.,Matsuda,K.,Takagi,M.&Yamamoto,N.(1997)
Detection of serum antibodies against phosphocholine-containing
aminoglycoglycerolipid specific to Mycoplasmafermentans in
HIV-1 infected individuals. J. Immunol. Methods 208, 103–113.
15. Seydel, U., Oikawa, M., Fukase, K., Kusumoto, S. & Branden-
burg, K. (2000) Intrinsic conformation of lipid A is responsible for
agonistic and antagonistic activity. Eur. J. Biochem. 267, 3032–3039.
16. Brandenburg,K.,Schromm,A.B.,Koch,M.H.J.&Seydel,U.
(1995) Conformation and fluidity of endotoxins as determinants
of biological activity. In Bacterial Endotoxins: Lipopolysaccharides
from Genes to Therapy (Levin, J., Alving, C.R., Munford, R.S. &
Redl, H., eds), pp. 167–182. John Wiley & Sons, New York.
17. Schromm, A.B., Brandenburg, K., Loppnow, H., Moran, A.P.,
Koch, M.H.J., Rietschel, E.Th & Seydel, U. (2000) Biological
activities of lipopolysaccharides are determined by the shape of
their lipid A portion. Eur. J. Biochem. 267, 2008–2013.
18. Bligh, E.G. & Dyer, W.J. (1959) A rapid method of total lipid
extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.
19. Galanos, C., Lu
¨
deritz, O. & Westphal, O. (1969) A new method
for the extraction of R. lipopolysaccharides. Eur. J. Biochem. 9,
245–249.
20. Mantsch, H.H. & McElhaney, R.N. (1991) Phospholipid phase
transitions in model andbiological membranes as studied by
infrared spectroscopy. Chem.Phys.Lipids57, 213–226.
21. Brandenburg, K., Funari, S.S., Koch, M.H.J. & Seydel, U. (1999)
Investigations into the acyl chain packing of endotoxins and
phospholipids under near physiological conditions by WAXS
and FTIR spectroscopy. J. Struct. Biol. 128, 175–186.
22. Kauppinen, J.K., Moffat, D.J., Mantsch, H.H. & Cameron, D.G.
(1981) Fourier self-deconvolution: a method for resolving
intrinsically overlapped bands. Appl. Spectrosc. 35, 271–276.
23. Brandenburg, K., Kusumoto, S. & Seydel, U. (1997) Con-
formational studies of synthetic lipid A analogues and partial
structures by infrared spectroscopy. Biochim. Biophys. Acta 1329,
193–201.
24. Brandenburg, K., Richter, W., Koch, M.H.J., Meyer, H.W. &
Seydel, U. (1998) Characterizationof the nonlamellar cubic and
HII structures of lipid Afrom Salmonella enterica serovar Min-
nesota by X-ray diffraction and freeze-fracture electron micro-
scopy. Chem. Phys. Lipids 91, 53–69.
25. Koch, M.H.J. & Bordas, J. (1983) X-ray diffraction and scattering
on disordered systems using synchrotron radiation. Nucl. Instr.
Meth. 208, 461–469.
26. Schromm, A.B., Brandenburg, K., Rietschel, E.T., Flad, H D.,
Carroll, S.F. & Seydel, U. (1996) Lipopolysaccharide binding
protein (LBP) mediates CD14-independent intercalation of lipo-
polysaccharide into phospholipid membranes. FEBS Lett. 399,
267–271.
27. Friberger, P., So
¨
rskog, L., Nilsson, K. & Kno
¨
s, M. (1987) The use
of a quantitative assay in endotoxin testing. Progr. Clin. Biol. Res.
231, 49–169.
28. Delude, R.L., Yoshimura, A., Ingalls, R.R. & Golenbock, D.T.
(1998) Construction ofa lipopolysaccharide reporter cell line and
its use in identifying mutants defective in endotoxin, but not
TNFa, signal transduction. J. Immunol. 161, 3001–3009.
29. Yoshimura, A., Lien, E., Ingalls, R.R., Tuomanen, E., Dziarski,
R. & Golenbock, D. (1999) Cutting edge: recognition of Gram-
positive bacterial cell wall components by the innate immune
system occurs via Toll-like receptor 2. J. Immunol. 163, 1–5.
30. Fringeli, U.P. & Gu
¨
nthard, H.H. (1981) Infrared membrane
spectroscopy. In Membrane Spectroscopy (Grell, E., ed.), pp.
270–332. Springer-Verlag, Berlin.
31. Brandenburg, K. & Seydel, U. (2002) Vibrational spectroscopy of
carbohydrates and glycoconjugates. In Handbook of Vibrational
Spectroscopy, Vol. 5 (Chalmers, J.M. & Griffiths, P.R., eds),
pp. 3481–3507. John Wiley & Sons, Chicester.
32. Mu
¨
hlradt, P.F. & Frisch, M. (1994) Purification and partial bio-
chemical characterizationofaMycoplasma fermentans-derived
substance that activates macrophages to release nitric oxide,
tumor necrosis factor, and interleukin-6. Infect. Immunol. 62,
3801–3807.
33. Calcutt, M.J., Kim, M.F., Karpas, A.B., Mu
¨
hlradt,P.F.&Wise,
K.S. (1999) Differential posttranslational processing confers
interspecies variation ofa major surface lipoprotein and a
macrophage-activating lipopeptide ofMycoplasma fermentans.
Infect. Immunol. 67, 760–771.
34. Takeuchi, O., Kaufmann, A., Grote, K., Kawai, T., Hoshino, K.,
Morr, M., Mu
¨
hlradt, P.F. & Akira, S. (2000) Cutting edge: pre-
ferentially the R-stereoisomer of the mycoplasmal lipopeptide
macrophage-activating lipopeptide-2 activates immune cells
through a toll-like receptor 2- and MyD88-dependent signaling
pathway. J. Immunol. 164, 554–557.
35. Schromm, A.B., Brandenburg, K., Loppnow, H., Za
¨
hringer, U.,
Rietschel, E.T., Carroll, S.F., Koch, M.H.J., Kusumoto, S. &
Seydel, U. (1998) The charge of endotoxin molecules influences
their conformation and interleukin-6 inducing capacity. J.
Immunol. 161, 5464–5471.
3278 K. Brandenburg et al. (Eur. J. Biochem. 270) Ó FEBS 2003
36. Brandenburg, K., Lindner, B., Schromm, A., Koch, M.H.J.,
Bauer,J.,Merkli,A.,Zbaeren,C.,Davies,J.G.&Seydel,U.
(2000) Physicochemical characteristics of triacyl lipid A partial
structure OM-174 in relation to biological activity. Eur. J. Bio-
chem. 267, 3370–3377.
37. Takeuchi, O. & Akira, S. (2001) Toll-like receptors; their physio-
logical role and signal transduction system. Int. Immunopharma-
col. 1, 625–635.
38. Kawahara, K., Seydel, U., Matsuura, M., Danbara, H., Rietschel,
E.T. & Za
¨
hringer, U. (1991) Chemical structure of glyco-
sphingolipids isolated from Sphingomonas paucimobilis. FEBS
Lett. 292, 107–110.
39. Krziwon, C., Za
¨
hringer, U., Kawahara, K., Weidemann, B.,
Kusumoto, S., Rietschel, E.T., Flad, H D. & Ulmer, A.J. (1995)
Glycosphingolipids from Sphingomonas paucimobilis induce
monokine production in human mononuclear cells. Infect.
Immunol. 63, 2899–2905.
40. Mu
¨
ller, M., Scheel, O., Lindner, B., Gutsmann, T. & Seydel, U.
(2002) The role of membrane-bound LBP, endotoxin aggregates,
and the MaxiK channel in LPS-induced cell activation.
J. Endotoxin Res. 9, 181–186.
41. Gutsmann, T., Mu
¨
ller, M., Carroll, S.F., MacKenzie, R.C.,
Wiese, A. & Seydel, U. (2001) Dual role of lipopolysaccharide
(LPS)-binding protein in neutralization of LPS and enhancement
of LPS-induced activation of mononuclear cells. Infect. Immunol.
69, 6942–6950.
42. Kirschning, C.J., : Wesche, H., Ayres, T. & Rothe, M. (1998)
Human toll-like receptor 2 confers responsiveness to bacterial
lipopolysaccharide. J. Exp. Med. 188, 2091–2097.
43. Beutler, B. (2000) TLR-4: central component of the sole mam-
malian LPS sensor. Curr. Opin. Microbiol. 3, 23–28.
44. Blunck, R., Scheel, O., Mu
¨
ller, M., Brandenburg, K., Seitzer, U. &
Seydel, U. (2001) New insights into endotoxin-induced activation
of macrophages: Involvement ofa K
+
channel in transmembrane
signalling. J. Immunol. 166, 1009–1015.
45. Ben-Menachem., G., Bystrom, T., Rechnitzer, H., Rottem, S.,
Rilfors, L. & Lindblom, G. (2001) The physico-chemical char-
acteristics of the phosphocholine-containing glycoglycerolipid
MfGl-II govern the permeability properties of Mycoplasma
fermentans. Eur. J. Biochem. 268, 3694–3701.
46. Cullis, P.R., de Kruijff, B., Hope, A.J., Verkleij, A.J., Nayar, R.,
Farren, S.B., Tı
´
lcock, C., Madden, T.D. & Bally, M.B. (1983)
Structural properties of lipids and their functional roles in bio-
logical membranes. In Membrane Fluidity in Biology: Concepts
of Membrane Structure, Vol. 1 (Aloia, R.C., ed.), pp. 39–81.
Academic Press, New York.
Ó FEBS 2003 Biophysical study ofMycoplasma glycolipid (Eur. J. Biochem. 270) 3279
. Physicochemical characterization and biological activity
of a glycoglycerolipid from
Mycoplasma fermentans
Klaus Brandenburg
1
, Frauke Wagner
1
, Mareike. Mu
¨
hlradt, P.F. & Frisch, M. (1994) Purification and partial bio-
chemical characterization of a Mycoplasma fermentans- derived
substance that activates macrophages