Koch2, Ulrich Za¨hringer1and Ulrich Seydel1 1 Forschungszentrum Borstel, Center for Medicine and Biosciences, Borstel;2European Molecular Biology Laboratory, Outstation Hamburg, Hamburg,
Trang 1Physicochemical characterization and biological activity
Klaus Brandenburg1, Frauke Wagner1, Mareike Mu¨ller1, Holger Heine1, Jo¨rg Andra¨1, Michel H J Koch2, Ulrich Za¨hringer1and Ulrich Seydel1
1
Forschungszentrum Borstel, Center for Medicine and Biosciences, Borstel;2European Molecular Biology Laboratory,
Outstation Hamburg, Hamburg, Germany
We report a comprehensive physicochemical
characteriza-tion of a glycoglycerolipid from Mycoplasma 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 Tc¼ 30 Cfor 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 of a 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 fermentansis 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 isolated two 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 biological activity 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 and biological activity of 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)
Trang 2Based 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, and from 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-factor-a) in humfactor-an mononuclefactor-ar cells, wherefactor-as 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 37Cas described
previously [12] After 68 h, cells were harvested, washed
twice with 0.25M NaCl in 10 mMTris/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 mM Tris/HCl
buffer, pH 7.5, containing 0.25M 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)1dried 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 HPLCon 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
2 mLÆmin)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,
Rf¼ 0.17 (yield 4.16 mg)
Lipid samples LPS from deep rough mutant Re from Salmonella minnesotastrain 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
Germany) Bovine brain 3-sn-PS and egg 3-sn-PCwere obtained from Sigma (Deisenhofen, Germany) For pre-paration of liposomes from a phospholipid mixture corresponding to the composition of the macrophage membrane (PLMN), PS or PCthe 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/Pi 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 60C, again vortexing, and recooling to 10C 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)2Mlipid 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 ms (CH2), which lies around 2850 cm)1in the gel and between
2852 cm)1 and 2853 cm)1in the liquid-crystalline phase [20,21]
Lipid headgroup conformation For a characterization of 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 CaF2 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 37C Usually, the original spectra were evaluated directly and a 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
Trang 3Aggregate 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 (s¼ 2 sin h/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 PCor 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 activity of 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 (21C, 500 g)
layer of mononuclear cells was collected and washed
three times in serum-free RPMI 1640 containing 2 mM
-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· 106mL)1 For stimulation, 200 mL per well heparinized MNC (5· 106mL)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 37Cand 5% CO2 Supernatants were collected after centrifugation of the culture plates for 10 min
at 400 g and stored at )20 Cuntil 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 4C For the 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 37Cin a humidified 5%
CO2 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· 105per 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 minnesota R595 are shown in Fig 1A,B, respect-ively MfGl-II and PCboth have a diacyl hydrophobic moiety and an identical phosphocholine headgroup Like LPS, MfGl-II carries two negatively charged phosphates
Trang 4and 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 ms
(CH2) revealed a phase transition at around 30Cfor
MfGl-II similar to that of deep rough mutant LPS from
S minnesotastrain 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
PCexhibits 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 (C¼ O), the
lipid scissoring band d (CH2) at 1465 cm)1, the
antisym-metric and symantisym-metric stretching vibrations of the negatively
charged phosphate groups mas(PO2) at 1220 cm)1and ms
(PO ) at 1120 cm)1, respectively [23,30], and the bands at
1172, 1085, and 1038 cm)1assigned to glucose ring vibrations [31] As no additional bands in the range of the amide vibrations centered at 1650 and 1550 cm)1can 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) of hydrated PC, MfGl-II, and L PS Re.
Trang 5lipopolysaccharides can be excluded The band contour of
mas(PO2) 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 PCshows the
occurrence of one major band around 1225 cm)1in
accordance with the well-known high water-binding
capa-city of lecithin headgroups [30] Finally, MfGl-II exhibits a
main band at 1245 cm)1and 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 PCliposomes 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 reflecdiffrac-tion 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 17.0¼ 8.48 2, 16.9¼ 6.90 6, 17.0 ¼ 4.90 12, 17.0 ¼ 3.47 24, which can be assigned to a cubic structure with a periodicity
aQ¼ (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 of a 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-II or L PS Re at t = 50 s and subsequent addition of LBP
(0.2 m M ) at t = 100 s in comparison to control NaCl/P i (phosphate
buffered saline) The concentration of the glycolipids, PCand PS was
10 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 of a 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).
Trang 6LAL 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, activity of 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 activity of 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 fermentanshas 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 characterization of 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 37C(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%.
Trang 7strong LBP-induced incorporation into negatively charged
phospholipid liposomes (Fig 4), and a diffraction pattern,
which is consistent with the existence of a 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 normal
(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
HPLCpurification 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‡1 mgÆmL)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 A and MfGl-II represent a mechanical disturbance leading to a conformational change of the protein and, with that, signal transduction
Recently, Ben-Menachem
physico-chemical characterization of 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-ted to range between 35 and 45C Furthermore, from
31P-NMR they proposed only lamellar phases as aggregate structure, which was deduced from the isotropic 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).
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