Báo cáo khoa học: Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity docx

Physicochemical characterization of carboxymethyl lipid A
derivatives in relation to biological activity
Ulrich Seydel
1
, Andra B. Schromm
1
, Lore Brade
1
, Sabine Gronow
1
,Jo
¨
rg Andra
¨
1
, Mareike Mu
¨
ller
1
,
Michel H. J. Koch
2
, Koichi Fukase
3
, Mikayo Kataoka
3
, Masaya Hashimoto
3
, Shoichi Kusumoto
3
and Klaus Brandenburg

1
1 Forschungszentrum Borstel, Leibniz-Zentrum fu
¨
r Medizin und Biowissenschaften, Borstel, Germany
2 European Molecular Biology Laboratory, c ⁄ o DESY, Hamburg, Germany
3 Osaka University, Department of Chemistry, Osaka, Japan
Lipopolysaccharide (LPS, endotoxin), as a major
amphiphilic component of the outer leaflet of the outer
membrane of Gram-negative bacteria, exerts in an
isolated form a variety of biological activities in mam-
mals [1]. Chemically, LPS consists of a hydrophilic
heteropolysaccharide, which is covalently linked to a
Keywords
endotoxic shock
2
; inflammation;
lipopolysaccharide; signal transduction
Correspondence
U. Seydel, Forschungszentrum Borstel,
Division of Biophysics Parkallee 10, D-23845
Borstel, Germany
Fax: +49 4537 188632
Tel: +49 4537 188232
E-mail:
(Received 6 September 2004, revised 3
November 2004, accepted 5 November
2004)
doi:10.1111/j.1742-4658.2004.04471.x
Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bac-
teria belongs to the most potent activators of the mammalian immune sys-

tem. Its lipid moiety, lipid A, the ‘endotoxic principle’ of LPS, carries two
negatively charged phosphate groups and six acyl chain residues in a
defined asymmetric distribution (corresponding to synthetic compound
506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the
two hydroxylated acyl chains, is agonistically completely inactive, but is a
strong antagonist to bioactive LPS when administered to the cells before
LPS addition. The two negative charges of lipid A, represented by the
two phosphate groups, are essential for agonistic as well as for antagonistic
activity and no highly active lipid A are known with negative charges other
than phosphate groups. We hypothesized that the phosphate groups could
be substituted by other negatively charged groups without changing the
endotoxic properties of lipid A. To test this hypothesis, we synthesized
carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-
CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their
physicochemical with their endotoxic properties. We found that, similarly
to compounds 506 and 406, also for their carboxymethyl derivatives a par-
ticular molecular (‘endotoxic’) conformation and with that, a particular
aggregate structure is a prerequisite for high cytokine-inducing capacity
and antagonistic activity, respectively. In other parameters such as acyl
chain melting behaviour, antibody binding, activity in the Limulus lysate
assay, and partially the binding of 3-deoxy-d-manno-oct-2-ulosonic acid
transferase
1
, strong deviations from the properties of the phosphorylated
compounds were observed. These data allow a better understanding of
endotoxic activity and its structural prerequisites.
Abbreviations
ATR, attenuated total reflectance; CM, carboxymethyl; EU, endotoxin unit; FRET, fluorescence resonance energy transfer; GlcN,
D-glucosamine; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; LAL, Limulus amebocyte lysate; LBP, lipopolysaccharide-binding protein;
LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MNC, mononuclear cells; NBD, 7-Nitrobenz-2-oxa-1,3-diazol-4-yl;

PE, phosphatidylethanolamine; Rh, rhodamine; TLR, Toll-like receptor; TNF, tumor necrosis factor.
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 327
hydrophobic lipid moiety, termed lipid A, which
anchors the molecule to the outer leaflet of the outer
membrane. Because free lipid A has been shown to be
responsible for the biological activity of LPS in most
in vitro and in vivo test systems, it has been termed the
‘endotoxic principle’ of endotoxin [2]. The specific
requirements for endotoxin to be biologically active
are still only partly defined. It has been shown, how-
ever, that for the full expression of biological activity,
lipid A must possess a particular chemical composition
and primary structure like that found in enterobacteri-
al strains. Thus, for example, lipid A from the biolo-
gically most potent LPS of the deep rough mutant
strain Escherichia coli F515 consists of a b-1,6-linked
d-glucosamine (GlcN) disaccharide carrying two negat-
ively charged phosphate groups in the 1- and 4¢-posi-
tions and six saturated fatty acids in a defined
asymmetric distribution – four at the nonreducing and
two at the reducing GlcN. Variation of this composi-
tion such as a reduction of the number of charges or
the number of acyl chains, a change in their distribu-
tion, or degree of saturation, results in a dramatic
reduction of biological activity [3,4]. These observa-
tions could be interpreted to indicate that a variation
of the primary structure of endotoxin molecules influ-
ences their physicochemical behaviour, and that this
physicochemical behaviour is correlated to the biologi-
cal activity.

Lipid A is an amphiphilic molecule and therefore
tends to form multimeric aggregates above a critical
concentration, which depends on its hydrophobicity.
The structures of lipid A aggregates were found to be
either nonlamellar inverted (cubic, Q or hexagonal,
H
II
) or lamellar (L) depending on the primary chemical
structure and the molecular shape of the composing
molecules
3
[5]. We have previously shown that a pecu-
liar molecular shape of the lipid A portion of endotoxin
is a prerequisite for the expression of endotoxic activ-
ity. Thus, hexaacyl lipid A (or synthetic compound
506), which adopts a nonlamellar cubic aggregate struc-
ture, with a conical molecular shape, is biologically
highly active [6]. Pentaacyl lipid A and tetraacyl lipid A
(synthetic compound 406) form lamellar structures,
which can be related to a cylindrical molecular shape,
and are agonistically inactive, but may be antagonistic,
i.e., block the action of agonistic LPS [6]. Furthermore,
we have reported that nonlipid A amphiphiles with cor-
responding conical shape and two phosphates may also
induce cytokines. This observation led us to propose a
‘generalized endotoxic principle’ [7].
For all these endotoxically active compounds, cell
activation takes place after binding to proteins such as
lipopolysaccharide-binding protein (LBP) and CD14
[8,9], which finally transport the compounds to integral

membrane proteins such as Toll-like receptor 4
(TLR4) [10] or the K
+
-channel MaxiK [11].
It has been reported that a prerequisite for endo-
toxic activity is a sufficiently high number of negative
charges, i.e., essentially the phosphate groups in the
lipid A region [12]. However, no systematic study on
the role of the kind of charges has been performed.
Thus, in the present study the phosphate groups in the
synthetic tetraacyl and hexaacyl compounds (406 and
506, respectively) were replaced by carboxymethyl
(CM) groups, and the corresponding compounds
(Bis-CM-406, CM-506, and Bis-CM-506; chemical
structures shown in Fig. 1) were characterized physico-
chemically and biologically. We have found that
important characteristics such as the aggregate struc-
tures and the cytokine-inducing properties remain
essentially unchanged or are modified only slightly.
Partially clear modifications are observed in the recog-
nition by serum and cell-surface binding proteins.
However, other important features like Limulus activ-
ity and antibody binding change upon replacement of
the phosphate groups by the carboxymethyl groups.
These findings allow a more general understanding of
endotoxin action.
Results
Phase transition behaviour and intramolecular
conformation
The b «a gel to liquid crystalline acyl chain melting

transition, the conformation and molecular orientation
of particular molecular groups with respect to the
Te traacyl lipid A (406)
Bis-CM-406
Hexaacyl lipid A (506)
CM - 506
Bis-CM-506
R
1
P
CH COOH
P
CH COOH
2
2
CH COOH
2
R
2
OH
OH
C-OH
C-OH
C-OH
12
12
12
R
3
OH

OH
C-OH
C-OH
C-OH
14
14
14
R
4
P
CH COOH
P
P
2
CH COOH
2
OH
O
O
O
NH
O
.
O
.
14 14
O
O
HO
NH

O
O
O
OH
.
14 14
O
OH
.
R
3
R
4
R
2
R
1
Fig. 1. Chemical structures of synthetic tetraacyl and hexaacyl lipid
A analogs. P, phosphate groups; R
1
–R
4
, side groups as indicated.
26
Characterization of carboxymethyl lipid A U. Seydel et al.
328 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS
membrane plane of the synthetic compounds were
investigated as potentially important determinants of
bioactivity [13].
In Fig. 2, the temperature dependence of the peak

position of the symmetric stretching vibrational band
v
s
(CH
2
) for the different compounds reveals a con-
siderable influence of the phosphate substitution in
the case of the tetraacyl compounds. The phase
transition temperature T
c
of compound 406 around
25 °C is shifted for compound Bis-CM-406 to 46 °C,
concomitantly the wavenumber values in the gel
phase decrease from around 2851 cm
)1
to lower than
2850 cm
)1
, indicating a higher state of order (lower
fluidity). In contrast, the T
c
values of the hexaacyl
compounds are very similar and lie slightly above
50 °C. The wavenumber values in the gel phase
decrease from 2850 cm
)1
for compound 506 to
2849.5 for CM-506 and to 2849.0 cm
)1
for Bis-CM-

506. The comparison of the different compounds at
the biologically relevant temperature 37 °C (vertical
line) shows the same sequence.
Infrared spectra in the wavenumber range 1800–
1500 cm
)1
of the amide and ester vibrational bands
are compared exemplarily for 406 and Bis-CM-406
(Fig. 3). The peak positions of the ester band contours
are around 1740–1742 cm
)1
and 1728–1731 cm
)1
,
respectively (obtained from the second derivative), and
thus similar for both. The bandwidth, however, for
Bis-CM-406 is smaller, indicating less mobility of the
ester groups than those of phosphorylated 406.
Importantly, also the amide I band, predominantly
resulting from C¼O stretching of the amide group, is
located at lower wavenumbers and is sharper, indica-
ting stronger water and ⁄ or cation binding and, with
that, higher order. Furthermore, the shoulder at 1600–
1607 cm
)1
for Bis-CM-406 should correspond to the
antisymmetric stretching of the negatively charged
carboxylate group [v
as
(COO

–
)], showing the molecule
in the charged state at neutral pH.
To determine the orientation of the diglucosamine
group with respect to the membrane plane, infrared
dichroic measurements on an attennuated total
reflectance (ATR) plate were performed with hydra-
ted lipid multilayers. The dichroic ratios, R, were
0 10203040506070
2849
2850
2851
2852
2853
2854
406
Bis-CM-406
506
CM-506
Bis-CM-506
Wavenumber (cm
–1
)
Temperature (C°)
Fig. 2. Gel to liquid crystalline phase behav-
ior of the hydrocarbon chains of the various
synthetic lipid A analogs. The peak position
of the symmetric stretching vibration of the
methylene groups v
s

(CH
2
) is plotted vs.
temperature. In the gel phase it is located
around 2850 cm
)1
, in the liquid crystalline
phase around 2852.5 to 2853.0 cm
)1
.
Fig. 3. Infrared spectra in the range 1800–1500 cm
)1
for com-
pounds 406 and Bis-CM-406, exhibiting the ester carbonyl band
around 1730 cm
)1
, the amide I band (predominantly C¼O stretch-
ing) in the range 1620–1660 cm
)1
, and amide II band (predomin-
antly N–H bending) around 1550 cm
)1
. The band around 1602 cm
)1
for compound Bis-CM-406 corresponds to the antisymmetric
stretching vibration of the the negatively charged carboxylate group
v
as
(COO
–

).
U. Seydel et al. Characterization of carboxymethyl lipid A
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 329
measured for the diglucosamine ring vibrational
bands at 1170 and 1045 cm
)1
, allowing calculation of
the inclination angle of the diglucosamine ring plane
with respect to the membrane plane [14]. In Table 1,
the data are summarized showing high R values for
the tetraacyl, corresponding to small inclination
angles (5–20°), and much smaller R values for the
hexaacyl compounds corresponding to high inclina-
tion angles (47–48°).
Supramolecular aggregate structures
The aggregate structure has been described to be an
essential determinant for the ability of endotoxins to
induce cytokines in immune cells [5]. We have applied
small-angle X-ray diffraction using synchrotron
radiation to elucidate the aggregate structure of Bis-
CM-406 and Bis-CM-506 (Fig. 4). For the tetraacyl
compound (Fig. 4A), in the temperature range 20–
80 °C only one sharp diffraction peak at 4.72 nm is
observed, which can be assigned to the periodicity of a
multilamellar stack. The diffraction pattern of the
hexaacyl compound (Fig. 4B) is more complex. At
40 °C, the main diffraction peak at 4.53 nm may be
assumed to result from a multilamellar aggregate. The
further reflections at 2.46, 1.94, and 1.16 nm, however,
belong to a different type of aggregate structure. Thus,

a superposition of a lamellar and a cubic phase is sug-
gested, because the relations 2.46 nmÆv5 ¼ 5.50 nm
and 1.94 nmÆv8 ¼ 5.50 nm hold. At 80 °C, the three
observable reflections are clearly indicative of an
inverted hexagonal H
II
structure, because 4.61 nm ¼
2.67 nmÆv3 and 4.61 nm ¼ 2.31 nmÆv4. This phase is
already observable at 60 °C (data not shown). Respect-
ive data for the phosphorylated compounds show only
(multi)lamellar structures for 406 [6], and a higher ten-
dency of compound 506 towards a cubic structure
(data not shown).
Incorporation into phospholipid cell membranes
As a prerequisite for agonistic as well as antagonistic
activity, the intercalation of endotoxins into target cell
membranes corresponding to the composition of the
macrophage membrane mediated by the LBP has been
described [15].
The results for the synthetic compounds are presen-
ted in Fig. 5, showing most pronounced intercalation
of the tetraacylated compound 406, and significantly
lower efficiencies for the other compounds.
Binding of monoclonal antibodies
The reactivities of several lipid A monoclonal antibod-
ies with compounds CM-506 and Bis-CM-506 were
compared to compound 506 using an ELISA. Selected
antibodies recognize variations in the hydrophilic
backbone as follows (Fig. 6A): the monoclonal anti-
bodies A6 and 8A1 recognize the bisphosphorylated

backbone, mAb S1 binds to the 4¢-monophosphory-
lated backbone and mAb A43 reacts with phosphoryl-
ated as well as with phosphate-free compounds [16].
As one example, the data of the phosphate-dependent
mAb A6 and of the phosphate-independent mAb A43
are shown in Fig. 6B, displaying antibody binding
curves determined by checkerboard titration. The
results show that both mAb’s bind with high affinity
to compound 506. mAb A43 binds with similarly high
affinities to compounds 506, CM-506, and Bis-CM-
506, whereas mAb A6 binds with high affinity only to
compound 506. Binding to compound CM-506 was
observed only at much higher antibody concentrations
and no binding was observed to Bis-CM-506. mAb
8A1 gave similar results as mAb A6 (data not shown).
mAb S1, which recognizes the 4¢-monophosphorylated
backbone, reacted not only with compound 506 but
also with compound CM-506 although with somewhat
lower affinity. No binding of mAb S1 to compound
Bis-CM-506 was observed.
Limulus amebocyte lysate (LAL)
The ability of the synthetic compounds to activate
the clotting cascade of the horseshoe crab Limulus
Table 1. Dichroic ratio R, order parameter S, and inclination angle h
between diglucosamine ring plane and membrane plane for the
synthetic lipid A analogs. The R values were evaluated from the
ratio of the band intensities of the 90° and 0° polarized infrared
spectra of the diglucosamine ring vibrations at 1045 and
1170 cm
)1

. The error of h results from calculating the Gaussian
error propagation by using the functional relation between R, S and
h [14].
Compound
Parameter
Dichroic
ratio R
Order
parameter S
Inclination
angle h (°)
406 1.33 0.55 20.4 ± 14.0
0.46 16.5 ± 18.2
0.33 7.1 ± 28.3
Bis-CM-406 1.60 0.69 15.0 ± 3.8
0.56 9.0 ± 7.1
0.49 4.9 ± 12.0
506 0.98 0.96 46.8 ± 1.3
CM-506 1.05 0.88 47.6 ± 1.6
Bis-CM-506 1.01 0.93 47.5 ± 1.2
Characterization of carboxymethyl lipid A U. Seydel et al.
330 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS
polyphemus gave highest values for compound 406,
followed by 506 and CM-506 (Table 2). Considering
that the diglucosamine 4¢-phosphate backbone is the
recognition structure of the LAL assay [17,18], it
seems surprising that compounds Bis-CM406 and Bis-
CM-506 also showed high reactivity down to
10 ngÆmL
)1

.
Cytokine-inducing capacity in macrophages
As a characteristic endotoxic reaction, the induction
of tumor necrosis factor a (TNFa) production in
human macrophages by the synthetic compounds was
determined. Concomitantly, the influence of the
specific MaxiK channel blocker paxilline on TNFa
production was monitored. Data are shown for com-
pounds 506 and Bis-CM-506 (Fig. 7) revealing cyto-
kine-inducing capacity down to 1 ngÆmL
)1
for the
former, whereas the activity of the latter is one order
of magnitude lower. The activity of compound
CM-506 is nearly the same as for compound 506
(data not shown). For both compounds inhibi-
tion due to the addition of paxilline can clearly
be observed, in particular at the lower lipid
log I
log I
0.2 0.4 0.6 0.8
Bis-CM-406
4.72 nm
20 °C
40
80
Bis-CM-506
1.16 nm
2.32 nm
2.46 nm

4.61 nm
1.94 nm
2.67 nm
4.53 nm
40 °C
80
s (nm
-1
)
A
B
Fig. 4. Synchrotron radiation small-angle
X-ray diffraction patterns for compounds
Bis-CM-406 (A) and Bis-CM-506 (B) at high
water content (90%) and different
temperatures.
50 100 150 200 250 300
0.5
1.0
1.5
2.0
2.5
+LBP
PL
liposomes
Buffer
CM-506
Bis-CM-506
506
Bis-CM-406

406
I
D
/I
A
Time (s)
Fig. 5. LBP-mediated intercalation of the
synthetic lipid A analogs into phospholipid
liposomes corresponding to the composition
of the macrophage membrane, derived from
the increase of the ratio of the donor fluor-
escence intensity, I
D
, to that of the accep-
tor, I
A
. At 50 s, the lipid A analogs were
added to the liposomes, and at 100 s LBP
was added.
U. Seydel et al. Characterization of carboxymethyl lipid A
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 331
concentrations, which is, however, significantly higher
for the CM- as compared to the phosphate-contaning
compound. This observation allows one to conclude
that channel blocking leads to inhibition of signal
transduction. Again, similar results are found for
CM-506.
GlcN II
GlcN I
P

P
O
O
N
N
14 1414 141214
OH
OH
S1
A43
A
B
A6, 8A1
Fig. 6. (A) Schematic representation of spe-
cificities of various monoclonal antibodies
against the lipid A backbone. The recogni-
tion structures of the mAbs A43, S1, and
A6 ⁄ 8A1 are GlcNII, diglucosamine-4¢-phos-
phate, and the entire backbone, respect-
ively. (B) Binding curves of monoclonal
antibodies A6 (left column) and A43 (right
column) to compounds 506 (top), CM-506
(middle), and Bis-CM-506 (bottom). ELISA
plates were coated with 400 (d), 200 (m),
100 (j), 50 (r), 25 (s), 12.5 (n), 6.3 (h)
and 3.1 (e) ng of compound per mL.
Antibody concentrations are indicated.
Values are the mean of quadruplicates with
confidence values not exceeding 10%.
Characterization of carboxymethyl lipid A U. Seydel et al.

332 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS
Antagonistic activity
Compound 406 is a well known effective antagonistic
agent against agonistically active LPS and lipid A [19].
The antagonistic action of compound Bis-CM-406 was
compared to that of 406 by the addition of these com-
pounds to mononuclear cells under serum-free condi-
tions 15 min prior to the addition of deep rough
mutant LPS in defined [antagonist] ⁄ [LPS] molar ratios.
Figure 8 shows a strong cytokine-inhibiting activity of
compound 406, which is also expressed, but to signifi-
cantly lesser extent by compound Bis-CM-406.
HEK cell system
Similar to compound 506, compound Bis-CM-506 acti-
vates HEK293 cells via TLR4 ⁄ MD2, but not via
TLR2 (Fig. 9). Thus, the change in the nature of
charges does not cause changes in the affinity to recep-
tors decisive for cell activation.
Reactivity with Kdo transferases
To assess the lipid A analogs CM-506 and Bis-CM-506
in comparison to compound 506 as acceptors for 3-de-
oxy-d-manno-oct-2-ulosonic acid (Kdo) transferases,
they were submitted to in vitro enzyme assays, and the
reaction products were detected with mAb A20, react-
ing with a terminal Kdo residue (Fig. 10). Kdo trans-
ferases from Haemophilus influenzae (monofunctional;
lane A) [20], E. coli (bifunctional, lane B) [21],
Table 2. Endotoxic activity in endotoxin unitsÆmL
)1
(EUÆmL

)1
)in
the chromogenic Limulus amebocyte lysate test for the synthetic
lipid A analogs at various concentrations. The data are representa-
tive of three independent sets of measurements. In the test,
14 EUÆmL
)1
corresponds to 1 ngÆmL
)1
of LPS from Escherichia coli
O55:B5.
Lipid
Concentration
1
lgÆmL
)1
100
ngÆmL
)1
10
ngÆmL
)1
1
ngÆmL
)1
100
pgÆmL
)1
406 > 125 > 125 > 125 45.84 1.96
Bis-CM-406 > 125 61.5 11.3 1.76 0.89

506 > 125 76.1 15.6 3.16
CM-506 > 125 23.6 11.8 2.20
Bis-CM-506 > 125 82.8 12.5 4.5 1.43
Fig. 8. Antagonistic activity of compounds 406 and Bis-CM-406.
Human mononuclear cells were incubated under serum-free condi-
tions with the antagonistic compound, and after 15 min LPS from
Salmonella minnesota R595 was added at the given molar ratios.
The data result from one representative experiment. The mean and
standard deviation are based on the data from the determination of
TNFa in duplicate at two different dilutions.
Fig. 7. Induction of TNFa production in human macrophages by
compounds 506 (A) and Bis-CM-506 (B) in the absence (left-hand
bars) and presence (right-hand bars) of the K
+
-channel blocker paxil-
line at different lipid concentrations. The concentration of paxilline
was 20 l
M.The data result from one representative experiment.
The mean and standard deviation are based on the data from the
determination of TNFa in duplicate at two different dilutions. A
repetition of the experiments yielded the same dependences
except for the absolute amount of TNFa production which may vary
significantly between different donors.
U. Seydel et al. Characterization of carboxymethyl lipid A
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 333
Burkholderia cepacia (bifunctional, lane C) [22], and
Chlamydia psittaci
4
(tetrafunctional, lane D) [23] were
tested for their ability to use the artificial lipid A ana-

logs as substrates. All Kdo transferases gave a product
when compound 506 served as acceptor, and the same
were observed when compound CM-506 was offered
as acceptor (data not shown).
5
However, compound
Bis-CM-506 was transformed to a Kdo-containing
product only when incubated with Kdo transferases
from H. influenzae or E. coli, but not with enzymes
from B. cepacia or C. psittaci. The use of monophos-
phoryl compound 504 (having the 4¢-phosphate) and
505 (1-phosphate) showed that both phosphate resi-
dues were necessary for binding of Kdo transferases
from B. cepacia and C. psittaci , whereas those from
E. coli and H. influenzae were reactive with each of the
monophosphoryl analogs (data not shown).
Discussion
The CM derivatives of lipid A exhibit in many aspects
comparable behavior to that of the phosphate-contain-
ing compounds. The substitution of the 1-phosphate by
CM has already been shown not to alter the basic cyto-
kine-inducing capacity of compound 506 [14], and this
is similarly true for the further substitution with the
4¢-CM group (Fig. 7B). There is, however, a significant
decrease of the activity in particular for the lower con-
centrations 10 and 1 ngÆmL
)1
(Fig. 7), which could be
confirmed in three independent experiments. The
observed aggregate structures may provide an explan-

ation for this difference (Fig. 4B and U Seydel, AB
Schromm, L Brade, S Gronow, J Andra
¨
,MMu
¨
ller,
MHJ Koch, K Fukase, M Kataoka, M Hashimoto,
S Kusumoto & K Brandenburg, unpublished data)
6
.
Compound Bis-CM-506 adopts a mixed multilamellar ⁄
zcubic aggregate structure at 40 °C. According to previ-
ous work, one main structural prerequisite for endo-
toxic activity is the existence of a pure cubic or a mixed
unilamellar ⁄ cubic aggregate structure of the lipid A
part of LPS [24], whereas multilamellar structures have
been shown to reflect inactive lipid A. Thus, the signifi-
cant amount of a multilamellar portion within the
aggregate may be responsible for the reduction of activ-
ity as compared to natural lipid A or synthetic com-
pound 506. Regarding the other prerequisites for
endotoxin activity, no significant difference between the
two compounds can be found. For example, the incli-
nation angle of the diglucosamine plane with respect to
the membrane plane is very similar (Table 1). Thus, the
interpretation is confirmed that the driving force for an
optimal packing of the acyl chains is their linkage and
distribution to the two glucosamines, causing the
observed high inclination independent of the type of
charges [14]. Also, all compounds show an LBP-

induced intercalation into phospholipid liposomes
(Fig. 5), for which the presence of negative charges is a
prerequisite. The presence of the infrared band around
1605 cm
)1
(Fig. 3) clearly indicates that the carboxylate
group is in the ionized state.
Analogously to the agonistically active hexaacyl
compounds is the antagonistic activity of the tetraacyl
compounds: Bis-CM-406 is also antagonistic, although
to a significantly lower degree than 406 (Fig. 8). Cor-
responding to the data of the antagonistic activity
0
500
1000
1500
2000
no Compound
506
Compound
Bis-CM-506
IL-1
Stimuli
Control
huTLR4/MD2
IL-8 concentration
(pg/ml)
TLR2/MD2
Fig. 9. Activation of HEK293 cells by compounds 506 and Bis-CM-
506 in dependence on TLR-expression. HEK293 cell were transi-

ently transfected with a control plasmid (pcDNA3), cotransfected
with expression plasmids for human TLR4 and human MD2 or for
human TLR2 as described in Materials and methods. After 24 h,
cells were stimulated with compounds 506, Bis-CM-506
(1 lgÆmL
)1
), or recombinant interleukin-1 (5 ngÆmL
)1
) for 24 h. Cell
activation was determined by measuring the IL-8 concentration in
an ELISA test. Transfections were performed in triplicate, and data
given are mean and standard deviation of one experiment represen-
tative of three.
506
CM-506
Bis-CM-506
406
A
C
D
E
B
Fig. 10. In vitro assays of different Kdo transferases using com-
pounds 506, CM-506, Bis-CM-506 and 406 as lipid acceptors. Reac-
tion products of Kdo transferases from H. influenzae (A), E. coli (B),
B. cepacia (C), C. psittaci (D) or controls without enzyme added (E)
were detected with mAb A20, recognizing a terminal Kdo residue.
Characterization of carboxymethyl lipid A U. Seydel et al.
334 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS
found for compound 406 [6], a pure multilamellar

aggregate structure is observed for Bis-CM-406
(Fig. 4A), and the inclination angle of the diglucosa-
mine ring plane with respect to the membrane plane is
small (Table 1). Furthermore, LBP mediates the inter-
calation of compound 406 into phospholipid mem-
branes more effectively than compound Bis-CM-406
(Fig. 5). This observation might be connected with the
much higher fluidity of compound 406 as compared
to compound Bis-CM-406: for these compounds the
behavior of the carboxymethyl derivatives is strongly
deviating from that of compounds substituted with
phosphate groups (Fig. 2). The replacement of phos-
phate by carboxymethyl leads to an overall decrease of
the wavenumbers of v
s
(CH
2
) and concomitantly an
increase in T
c
(Fig. 2), i.e., a considerable ordering of
the hydrophobic moiety takes place. This observation
may be understood in the light of a dramatically
increased water ⁄ cation binding of the interface as
deduced from the red shift of the amide I band
(Fig. 3), which is caused by the presence of the carb-
oxymethyl groups.
These data are in accordance with the fact that the
most potent antagonists described so far [25,26] have
an extremely high fluidity. This holds for lipid A from

Rhodobacter capsulatus and Rhodobacter sphaeroides as
well as for lipid A from Chromobacterium violaceum
([5] and U Seydel, AB Schromm, L Brade, S Gronow,
J Andra
¨
,MMu
¨
ller, MHJ Koch, K Fukase, M Kata-
oka, M Hashimoto, S Kusumoto & K Brandenburg,
unpublished data)
7
.
The results from the LAL test for the bioactivity
(Table 2) are fundamentally different from those for
the cytokine-inducing capacity in human cells,
because in the former the tetra- and hexaacyl lipid A
CM-analogs exhibit similar activities except for minor
modifications, in that the biscarboxymethylated com-
pounds exhibit a lower activity than the phosphate-
containing compounds. This may be understood by
considering previous findings that the main epitope in
the Limulus test is the GlcNII-4¢-phosphate region
in the lipid A backbone [17,18]. A substitution of the
4¢-phosphate by another charge does not lead to a
complete inhibition, which implies that the Limulus
test recognizes patterns rather than a particular
charged group.
The binding specificities of the monoclonal antibod-
ies A6, 8A1, S1, and A43 are determined by the phos-
phorylation pattern [16]. So far it has not be

determined whether phosphate groups are specifically
required or whether they may be replaced by other
negatively charged groups. By synthesizing compounds
CM-506 and Bis-CM-506, we are in the position to
answer this question. The binding of mAb A43, which
recognizes the nonreducing GlcN moiety in the back-
bone of compound 506 is not influenced by the
replacement of phosphate groups by CM residues. This
is in agreement with the known specificity of mAb
A43, which does not require the phosphate substitu-
tion in either position. The binding of mAb A6 and
8A1 to compound CM-506 (for the latter, data not
shown) is considerably reduced as compared to com-
pound 506 (Fig. 6). This result is also not unexpected
because previous data had already shown that binding
of both mAbs to 4¢-monophosphorylated lipid A
required 30-fold higher antigen concentrations than
those needed with bisphosphorylated lipid A [16]. No
binding of mAbs A6 and 8A1 was observed with Bis-
CM-506 showing that the bisphosphorylated backbone
is a prerequisite for high affinity binding.
In summary, the data confirm that lipid A antibod-
ies, except mAb A43, recognize a distinct phosphoryla-
tion pattern of the lipid A backbone and confirm that
phosphates are part of the epitopes recognized, which
cannot be replaced simply by other negatively charged
groups. This may be understood by different negative
charge densities within the phosphate and the carboxy-
methyl groups.
Recently, it has been described that a blockade of

the K
+
-channel, MaxiK, by the specific blocker paxil-
line is connected to an inhibition of cytokine induc-
tion in macrophages [11]. The test of the synthetic
hexaacyl compounds showed that although Bis-CM-
506 is less effective than 506 in the cytokine assay,
the action of the K
+
-channel (MaxiK) blocker paxil-
line is reversed: our data indicate a higher efficiency
in the case of the carboxylated compound; a complete
blockade of the cytokine induction is observed
already at 100 ngÆmL
)1
for Bis-CM-506 (Fig. 7).
Together with the data of the activation of HEK cells
(Fig. 9), which indicate the involvement of the
TLR4 ⁄ MD2 complex in cell activation, these data
provide clear evidence that not a single molecular
species but rather a complete receptor cluster [27]
governs cell signalling.
Interestingly, both lipid A analogs show different
properties when used as acceptors for Kdo transfer-
ases. Whereas the enzymes from H. influenzae and
E. coli are not depending on either phosphate residue
substituting the lipid A backbone and recognize both
compounds CM-506 and Bis-CM-506 as acceptors,
the respective enzymes from B. cepacia and C. psittaci
are strongly depending on the phosphate group in

position 4¢. Both Kdo transferases accept compound
CM-506 but are not able to recognize compound Bis-
CM-506 as a Kdo acceptor. Presently, the reason for
U. Seydel et al. Characterization of carboxymethyl lipid A
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 335
this distinctive behavior of Kdo transferases cannot
be explained completely, in particular with respect to
the similarity of the molecular conformations of
compounds 506 and Bis-CMz-506 (Table 1). How-
ever, together with the data of the monophosphoryl
lipid A compounds a necessary prerequisite for the
binding of Kdo transferases is the existence of a
charge at position 4¢. That two of the transferases
bind to both compounds 506 as well as Bis-CM-506
and the other two do not, may have to do with the
acyl chain substitution of the acceptor, which again
may play a decisive role for the molecular conforma-
tion. It was shown that LPS from H. influenzae sim-
ilar to that from E. coli is hexaacylated [21], whereas
those from B. cepacia [23] and C. psittaci [28] are
pentaacylated.
Conclusions
The mere presence of two negative charges but not their
kind within the lipid A backbone is essential for the
bioactivity of endotoxins, for the agonistic as well as
the antagonistic activities. These findings confirm and
extend our ‘conformational concept’ of endotoxicity,
presuming for agonists a conical shape of the lipid A
moiety with a high inclination angle of the acyl chains
with respect to the membrane surface and for antago-

nists a cylindrical shape with a low inclination angle.
For other effects, however, for which particular binding
epitopes are necessary, exchange of charges may largely
change the bioactivities. This is valid for the recognition
by monoclonal antibodies, the binding of Kdo trans-
ferases, and the reactivity in the Limulus test.
Materials and methods
Synthesis
The synthesis of hexaacyl lipid A compounds 506 [29,30],
CM-506 [30], and tetraacyl lipid A 406 [31,32] has been pub-
lished previously. Monophosphoryl compounds 504
(4¢-phosphate) and 505 (1-phosphate), used in some cases,
were synthesized as described [29]. The synthesis of Bis-CM-
506 and Bis-CM-406 will be reported elsewhere (K Fukase,
M Kataoka, M Hashimodo & S Kusumodo, unpublished
data).
8
Briefly, the 3-position of 1-O-allyl 4,6-O-benzylidene-
2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-a-d-gluco-
pyranoside was acylated with (R)-3-benzyloxytetradecanoic
acid. The 2-N-Troc (Troc ¼ 2,2,2-trichloroethoxycarbonyl)
group was cleaved, and the 2-amino group was then acylated
with (R)-3-benzyloxytetradecanoic acid. The allyl group was
then oxidized with OsO
4
, and the resulting diol was oxida-
tively cleaved with Pb(OAc)
4
to give 1-O-formylmethyl
glycoside, which was further oxidized with NaClO

2
. The
resulting carboxyl group was protected with benzyl group by
using phenyldioazomethane. Deprotection of benzylidene
gave the reducing-end GlcN moiety as a common glycosyl
acceptor for the synthesis of Bis-CM-506 and Bis-CM-406.
For the synthesis of the nonreducing-end GlcN moiety,
the common intermediate 1-O-allyl 4,6-O-benzylidene-3-
O-((R)-3-benzyloxytetradecanoyl)-2-deoxy-2-(2,2,2-trichloro-
ethoxy carbonylamino)-a-d-glucopyranoside was treated
under the conditions of regioselective reduction of benzylid-
ene (BF
3
ÆOEt
2
9
and Et
3
SiH) to give the 6-O-benzyl-4-OH
GlcN derivative. The benzyloxy carbonyl group was then
introduced to the 4-O-position using Ag
2
O and ICH
2
COO-
Bn. The 1-O-allyl group was then cleaved and transformed
to 1-O-trichloroacetimidate, which was used as a glycosyl
donor for the synthesis of Bis-CM-406. Glycosylation of
the above glycosyl acceptor with the glycosyl donor gave
the desired a(1-6) disaccharide. The 2¢-N-Troc group was

cleaved, and the resulting amino group was acylated with
(R)-3-benzyloxytetradecanoic acid to give the protected Bis-
CM-406. The final catalytic hydrogenation gave the desired
Bis-CM-406 (m ⁄ z ¼ 1360.0 [(M-H)
–
]).
For the synthesis of Bis-CM-506, the benzyl group of
the benzyloxytetradecanoyl moiety in the above synthetic
intermediate 1-O-allyl 6-O-benzyl-4-O-benzyloxycarbonyl-
methyl-3-O-((R)-3-benzyloxytetradecanoyl)-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-a-d-glucopyra-noside was
selectively cleaved using 2,3-dichloro-5,6-dicyanobenzoqui-
none. The resulting hydroxy group of the fatty acid moi-
ety was then acylated with tetradecanoic acid. After
deprotection of the 1-O-allyl group and formation of 1-O-
trichloroacetoimidate, glycosylation of the acceptor with
1-O-trichloroacetoimidate gave the disaccharide, which was
then transformed to Bis-CM-506 in a similar manner
(m ⁄ z ¼ 1753.1 [(M-H)
–
]).
The chemical structures of the synthesized compounds
are plotted in Fig. 1.
Lipids and reagents
Lipopolysaccharide from the deep rough mutant Escheri-
chia coli strain WBB01 (kindly provided by W Brabetz,
Biomet, Dresden, Germany)
10
was extracted by the phe-
nol ⁄ chloroform ⁄ petrol ether method [33] from bacteria

grown at 37 °C, purified, and lyophilized. Paxilline was
purchased from Sigma (Deisenhofen, Germany). LBP was a
kind gift of RL Dedrick (XOMA Co., Berkeley, CA,
USA). LBP was stored at ) 70 °Casa1mgÆmL
)1
stock
solution in 10 mm Hepes, pH 7.5, 150 mm NaCl, 0.002%
(v ⁄ v) Tween 80, 0.1% F68.
The lipids 3-sn-phosphatidylserine, egg 3-sn-phosphatidyl-
choline, sphingomyelin from bovine brain, and 3-sn-phos-
phatidylethanolamine from E. coli were from Avanti Polar
Lipids (Alabaster, AL, USA).
Characterization of carboxymethyl lipid A U. Seydel et al.
336 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS
Preparation of lipid aggregates
The synthetic lipid A analogs were dispersed in 20 mm
Hepes (pH 7) buffer (lipid concentration 0.01–10 mm for
the physical techniques and down to 100 pgÆ mL
)1
for the
biological assays), vortexed, sonicated for 30 min, and sub-
jected to several temperature cycles between 20 and 60 °C.
Finally, the lipid suspensions were incubated at 4 °C for at
least 12 h before use.
For preparation of liposomes corresponding to the com-
position of the macrophage membrane, phosphatidyl-
choline, phosphatidylserine, phosphatidylethanolamine and
sphingomyelin in a molar ratio of 1 : 0.4 : 0.7 : 0.5 [12]
were solubilized in chloroform, the solvent was evaporated
under a stream of nitrogen, the lipids were resuspended in

the appropriate volume of buffer, and treated as described
above for the synthetic lipid A analogs.
FTIR spectroscopy
The infrared spectroscopic measurements were performed
on an IFS-55 spectrometer (Bruker, Karlsruhe, Germany).
For measurements of the gel to liquid crystalline phase
transition, the lipid samples were placed in a CaF
2
cuvette
with a 12.5 lm Teflon spacer. Temperature scans were per-
formed automatically between 10 and 70 °C with a heating
rate of 0.6 ° CÆmin
)1
. For measurement of hydrated sam-
ples, the lipid samples were spread on an ATR Ge plate,
and free water was evaporated. Every 3 °C, 50 interfero-
grams were accumulated, apodized, Fourier transformed,
and converted to absorbance spectra.
Attenuated total reflectance (ATR) with polarized
infrared light
The lipids were prepared as hydrated oriented thin
multilayers as described previously [14] by spreading a
1mm lipid suspension in 20 mm Hepes buffer (pH 7)
on a ZnSe ATR crystal and evaporating the excess water
by slow periodic movement under a nitrogen stream at
room temperature. The lipid sample was placed in a
closed cuvette, and the air above the sample was satur-
ated with water vapour to maintain full hydration. Infra-
red ATR polarized spectra at 0 and 90° were recorded
using a mercury-cadmium-telluride detector with a scan

number of 1000 at a resolution of 2 cm
)1
. The measure-
ments were performed at 28 °C, the intrinsic instrument
temperature.
Dichroic ratios, R, for particular vibrational bands from
the diglucosamine backbone were evaluated at 1170 and
1045 cm
)1
. The angle h represents the angle between the
diglucosamine backbone and the direction of the hydrocar-
bon chains, and can be calculated from the measured R, if
an approximate value of the order parameter, S, is known.
As a first approximation, the S value calculated according
to a relationship between S, h and R as reported previously
[14], was used.
X-ray diffraction
X-ray diffraction experiments to define aggregate structures
of the lipid A analogues were performed at the European
Molecular Biology Laboratory (EMBL) outstation at the
Hamburg synchrotron radiation facility HASYLAB using
the double-focussing monochromator-mirror camera X33
[34]. X-ray diffraction patterns, obtained with exposure
times of 2 min using a linear gas proportional detector with
delay line readout [35], were evaluated according to previ-
ously described procedures [24]. These allow one to assign
the spacing ratios to defined three-dimensional aggregate
structures, from which the conformation of the individual
molecules can be deduced.
Fluorescence resonance energy transfer (FRET)

spectroscopy
Transport of the lipids into liposomes made from a phos-
pholipid mixture corresponding to that of the macrophage
membrane, mediated by LBP was determined by FRET
spectroscopy using the dyes 7-Nitrobenz-2-oxa-1,3-diazol-
4-yl (NBD)
11
phosphatidylethanolamine (NBD-PE) and rhod-
amine (Rh)-PE. The FRET assay was performed as a probe
dilution assay as described previously [12,15]. Briefly, phosp-
holipid liposomes corresponding to the composition of the
macrophage membrane were doubly labeled with NBD-PE
and Rh-PE (Molecular Probes, Eugene, OR, USA). Interca-
lation of unlabeled molecules into the doubly labeled lipo-
somes leads to probe dilution, thus inducing a lower FRET
efficiency: the emission intensity of the donor I
D
increases
and that of the acceptor I
A
decreases (for clarity, only the
quotient of the donor and acceptor emission intensity is
shown here). To the doubly labeled liposomes first the lipids
and then LBP were added, all at a final c oncentration of 10 lm.
Biological assays
12
Stimulation of macrophages
13
Monocytes were isolated from peripheral blood taken from
healthy donors by the Hypaque–Ficoll density gradient

method. Experiments were undertaken with the understand-
ing and written consent of each subject. To differentiate the
monocytes from the macrophages, cells were cultivated in
Teflon bags in the presence of 2 ngÆmL
)1
macrophage
colony-stimulating factor (M-CSF)
14
in RPMI 1640 medium
(endotoxin < 0.01 endotoxin units (EU)ÆmL
)1
in Limulus
test; Biochrom, Berlin, Germany) containing 2 mml-gluta-
mine, 100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin,
and 4% heat-inactivated human serum type AB at 37 °C and
6% CO
2
. On day 6 the cells were washed with NaCl ⁄ P
i
,
U. Seydel et al. Characterization of carboxymethyl lipid A
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 337
detached by trypsin ⁄ EDTA treatment and seeded at 1 · 10
5
per mL
15

in complete medium in 96 well tissue culture plates
(NUNC, Wiesbaden, Germany). After stimulation of the
cells with the compounds 506 and Bis-CM-506 for 4 h, cell-
free supernatant of duplicate samples were collected, pooled
and stored at ) 20 °C until determination of cytokine content.
Immunological determination of TNFa in the cell super-
natant was performed in a sandwich-ELISA as described
before [36]. Monoclonal mouse anti-human TNFa IgG
16;17
(clone 16 from Intex AG
16;17
, Muttenz, Switzerland) was used
to coat 96 well plates (Greiner, Solingen, Germany). Cell
culture supernatants and the standard [recombinant human
TNFa (rTNFa); Intex AG] were diluted with buffer. After
exposure to appropriately diluted test samples and serial
dilutions of standard rTNFa, the plates were exposed to
peroxidase-conjugated sheep anti-TNFa IgG. Subsequently,
the color reaction was started by addition of tetram-
ethylbenzidine ⁄ H
2
O
2
in alcoholic 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 was measured photometrically on an ELISA reader
at a wavelength of 450 nm and the values were related to
the standard. TNFa was determined in duplicate at two dif-
ferent dilutions and the values were averaged.

To study the influence of the K
+
-channel (MaxiK) on
cytokine induction, the specific channel blocker paxilline
was added at a concentration of 20 lm 10 min before sti-
mulation by the synthetic compounds to the mononuclear
cells, and incubated at 37 °C.
Antagonism
Mononuclear cells (MNC) were isolated from peripheral
blood taken from healthy donors by the Hypaque–Ficoll
density gradient method. The cell number was equilibrated
at 5 · 10
6
mL
)1
18
RPMI 1640 containing 2 mml-glutamine,
100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin. For
stimulation, 200 lLÆwell
)1
MNC were transferred into
96 well culture plates. The stimuli were serially diluted in
RPMI 1640 and added to the cultures at 20 lLÆwell
)1
.To
investigate the antagonistic effect the compounds 406 and

Bis-CM-406 had on LPS-induced TNFa production, the
antagonists were added to the cells 15 min prior to stimula-
tion with deep rough mutant LPS. The cultures were
incubated for 4 h at 37 °C under 5% CO
2
. Cell-free supern-
atants were collected after centrifugation of the culture
plates for 10 min at 400 g and stored at )20 °C until deter-
mination of cytokine content (see above).
Determination of endotoxin activity by the
chromogenic Limulus test
Endotoxin activity of the lipid A analogs was determined
by a quantitative kinetic assay based on the reactivity of
Gram-negative endotoxin with Limulus amebocyte lysate
(LAL) at 37 °C, using test kits of LAL Coamatic Chromo-
LAL K (Chromogenix, Haemochrom Milano, Italy)
19
([37]).
The standard endotoxin used in this test was from E. coli
(O55:B5), and 10 EUÆmL
)1
correspond to 1 ngÆmL
)1
.In
this assay, saturation occurs at 125 EUÆmL
)1
, and the reso-
lution limit is > 0.1 EUÆmL
)1
(maximum value for ultra-

pure water; Aqua B. Braun, Brann Melsungen, Germany)
20
.
Activation of HEK293 cells
HEK293 cells were transiently transfected using the follow-
ing protocol. For transfection, 0.2 lg of the indicated plas-
mids were diluted in 30 lL serum-free DMEM medium
(Qiagen, Hilden, Germany) containing 2 mml-glutamine,
100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin, added
to the wells of a 96 well dish, and precipitated by addition of
polyfect (1 lLin20lL serum-free DMEM) and incubation
for 10 min at room temperature. Subsequently, HEK293
cells were directly seeded onto the precipitates at a density of
7.5 · 10
4
cells per well and incubated overnight at 37 °C and
5% (v ⁄ v) CO
2
. The following day, cells were stimulated with
the indicated amounts of the compounds 506 and Bis-
CM-506, or recombinant human IL-1 (Peprotech, Rocky
Hill, NJ, USA) for 24 h. Cell activation was determined by
measuring IL-8 content in cell-free supernatants by using the
human IL-8 Cytoset antibody pair for ELISA (Bioscource,
Nivelles, Belgium), exactly according to the manufacturer’s
protocol. Transient transfections were carried out in tripli-

cate and data given are the mean and standard deviation.
Binding of monoclonal antibodies
For the analysis of the binding of antibodies to the back-
bone of lipid A, monoclonal antibodies with different epi-
tope specificities (Fig. 6A) were applied. The antibodies
raised against lipid A were produced as described earlier
[38]. The synthetic lipid A compounds were used in an
ELISA as solid-phase antigen as described [20]. For this,
polyvinyl plates (Falcon
21
3911, BD Biosciences, Franklin
Lakes, NJ, USA) were coated with various amounts of lipid
A dissolved in NaCl ⁄ P
i
(10 mm pH 7.3, 0.9% NaCl, 50lL)
at 4 °C overnight. All following steps were performed at
37 °C with gentle agitation, all washing steps were per-
formed four times. Coated plates were washed in NaCl ⁄ P
i
,
treated for 1 h with blocking buffer (2.5% casein in
NaCl ⁄ P
i
) and then incubated for 1 h with mAb diluted in
blocking buffer (50 lL). Plates were washed in NaCl ⁄ P
i
and incubated for 1 h with peroxidase-conjugated goat
anti-mouse IgG (heavy and light chain specific, (Dianova
Hamburg, Germany)
22

; diluted 1 : 1000 in blocking buf-
fer, 50 lL). After three washes in NaCl ⁄ P
i
, the plates
were washed in substrate buffer (0.1 m sodium citrate,
pH 4.5). Substrate solution, which was prepared freshly,
was composed of azinodi-3-ethylbenzthiazolinsulfonic acid
(1 mg) dissolved in substrate buffer (1 mL) sonicated in an
Characterization of carboxymethyl lipid A U. Seydel et al.
338 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS
ultrasound water bath for 3 min followed by the addition
of H
2
O
2
(25 lL of a 0.1% solution). After 30 min, the reac-
tion was stopped by addition of 2% aqueous oxalic acid
and the plates were read with a microplate reader (Dyna-
tech
23
MR5000, Dynatech Laboratories, Chantilly, VA,
USA) at a wavelength of 405 nm.
All tests were set up in quadruplicate. Confidence values
of the means were less than 10%.
Binding of Kdo transferases
Cell-free extracts containing Kdo transferase from E. coli,
C. psittaci or H. influenzae were prepared from Corynebacte-
rium glutamicum strains R163 ⁄ pJKB16, R163 ⁄ pJKB17 and
R163 ⁄ pCB23, respectively, as described previously [39].
From B. cepacia, crude cell-free extracts were used. The in vi-

tro tests were carried out using the bisphosphoryl compounds
506, CM-506 and Bis-CM-506 and monophosphoryl com-
pounds 504 and 505 as lipid acceptors as described elsewhere
[22], reaction mixtures were incubated for 1 h at 37 °C and
stopped by freezing at )20 °C. Aliquots were spotted on
nitrocellulose membranes and immunological identification
of the reaction products was performed as described [21]
using mAb A20, recognizing a terminal Kdo residue [23].
Acknowledgements
We are indebted to G. von Busse, C. Hamann, U.
Diemer, N. Hahlbrock, and K. Stephan for technical
assistance in the IR spectroscopic, FRET, LAL, and
cytokine measurements, respectively. We thank D.
Golenbock (Harvard Medical School, Boston, MA,
USA) for supplying us with expression plasmids for
human TLR2 and TLR4, K. Miyake (University of
Tokyo, Japan) for supplying the expression plasmid
for human MD2, and R. Dedrick (XOMA Co, Berke-
ley, CA, USA) for the kind gift of LBP.
This work has been carried out with financial sup-
port of the Deutsche Forschungsgemeinschaft (SFB
367, project B8), and from the Commission of the
European Communities, specific RTD programme
‘Quality of Life and Management of Living Resources’,
QLK-CT-2002-01001, ‘Antimicrobial endotoxin neut-
ralizing peptides to combat infectious diseases’.
References
1 Alexander C & Rietschel ET (2001) Bacterial lipopoly-
saccharides and innate immunity. J Endotoxin Res 7,
167–202.

2 Rietschel ET, Brade H, Holst O, Brade L, Mu
¨
ller-Loen-
nies S, Mamat U, Za
¨
hringer U, Beckmann F, Seydel U,
Brandenburg K, Ulmer AJ, Mattern T, Heine H,
Schletter J, Hauschildt S, Loppnow H, Scho
¨
nbeck U,
Flad H-D, Schade UF, Di Padova F, Kusumoto S &
Schumann RR (1996) Bacterial endotoxin: Chemical
constitution, biological recognition, host response, and
immunological detoxification. Curr Top Microbiol
Immunol 216, 39–81.
3Za
¨
hringer U, Lindner B & Rietschel ET (1994) Mole-
cular structure of lipid A, the endotoxic center of bac-
terial lipopolysaccharides. Adv Carbohydr Chem
Biochem 50, 211–276.
4 Raetz CR (1990) Biochemistry of endotoxins. Annu Rev
Biochem 59, 129–170.
5 Brandenburg K, Mayer H, Koch MHJ, Weckesser J,
Rietschel ET
24
& Seydel U (1993) Influence of the supra-
molecular structure of free lipid A on its biological
activity. Eur J Biochem 218, 555–563.
6 Schromm AB, Brandenburg K, Loppnow H, Moran

AP, Koch MHJ, Rietschel ET & Seydel U (2000) Biolo-
gical activities of lipopolysaccharides are determined by
the shape of their lipid A portion. Eur J Biochem 267,
2008–2013.
7 Seydel U, Hawkins L, Schromm AB, Heine H, Scheel
O, Koch MH & Brandenburg K (2003) The generalized
endotoxic principle. Eur J Immunol 33, 1586–1592.
8 Hailman E, Lichenstein HS, Wurfel MM, Miller DS,
Johnson DA, Kelley M, Busse LA, Zukowski MM &
Wright SD (1994) Lipopolysaccharide (LPS)-binding
protein accelerates the binding of LPS to CD14. J Exp
Med 179, 269–277.
9 Tobias PS, Soldau K, Gegner JA, Mintz D & Ulevitch
RJ (1995) Lipopolysaccharide binding protein-mediated
complexation of lipopolysaccharide with soluble CD14.
J Biol Chem 270, 10482–10488.
10 Chow JC, Young DW, Golenbock DT, Christ WJ &
Gusovsky F (1999) Toll-like receptor-4 mediates lipo-
polysaccharide-induced signal transduction. J Biol Chem
274, 10689–10692.
11 Blunck R, Scheel O, Mu
¨
ller M, Brandenburg K, Seitzer
U & Seydel U (2001) New insights into endotoxin-
induced activation of macrophages: Involvement of a
K
+
channel in transmembrane signaling. J Immunol
166, 1009–1015.
12 Schromm AB, Brandenburg K, Loppnow H, Za

¨
hringer
U, Rietschel ET, Carroll SF, Koch MHJ, Kusumoto S
& Seydel U (1998) The charge of endotoxin molecules
influences their conformation and interleukin-6 inducing
capacity. J Immunol 161, 5464–5471.
13 Seydel U, Wiese A, Schromm AB & Brandenburg K
(1999) A biophysical view on the function and activity
of endotoxins. In Endotoxin in Health and Disease
(Morrison D, Brade H, Opal S & Vogel S, eds), pp.
195–220. Marcel Dekker, New York.
14 Seydel U, Oikawa M, Fukase K, Kusumoto S & Bran-
denburg K (2000) Intrinsic conformation of lipid A is
responsible for agonistic and antagonistic activity. Eur J
Biochem 267, 3032–3039.
U. Seydel et al. Characterization of carboxymethyl lipid A
FEBS Journal 272 (2005) 327–340 ª 2004 FEBS 339
15 Gutsmann T, Schromm AB, Koch MHJ, Kusumoto S,
Fukase K, Oikawa M, Seydel U & Brandenburg K
(2000) Lipopolysaccharide-binding protein-mediated
interaction of lipid A from different origin with phos-
pholipid membranes. Phys Chem Chem Phys 2, 4521–
4528.
16 Kuhn H-M, Brade L, Appelmelk BJ, Kusumoto S,
Rietschel ET & Brade H (1992) Characterization of the
epitope specificity of murine monoclonal antibodies
directed against lipid A. Infect Immun 60, 2201–2210.
17 Brandenburg K, Ju
¨
rgens G, Andra

¨
J, Lindner B, Koch
MHJ, Blume A & Garidel P (2002) Biophysical charac-
terization of the interaction of high-density lipoprotein
(HDL) with endotoxins. Eur J Biochem 269, 5972–5981.
18 Takada H, Kotani S, Tanaka S, Ogawa T, Takahashi I,
Tsujimoto M, Komuro T, Shiba T, Kusumoto S &
Kusunose N (1988) Structural requirements of lipid A
species in activation of clotting enzymes from the horse-
shoe crab, and the human complement cascade. Eur J
Biochem 175, 573–580.
19 Kovach NL, Yee E, Munford RS, Raetz CRH & Har-
lan JM (1990) Lipid IVa inhibits synthesis and release
of tumor necrosis factor induced by lipopolysaccharide
in human whole blood ex vivo. J Exp Med 172, 77–84.
20 Mu
¨
ller-Loennies S, Brade L & Brade H (2002) Chemical
structure and immunoreactivity of the lipopolysacchar-
ide of the deep rough mutant I-69 Rd- ⁄ b+ of Haemo-
philus influenzae. Eur J Biochem 269, 1237–1242.
21 Gronow S, Brabetz W & Brade H (2000) Comparative
functional characterization in vitro of heptosyltransfer-
ase I (WaaC) and II (WaaF) from Escherichia coli. Eur
J Biochem 267, 6602–6611.
22 Gronow S, Noah C, Blumenthal A, Lindner B & Brade
H (2003) Construction of a deep-rough mutant of Bur-
kholderia cepacia ATCC 25416 and characterization of
its chemical and biological properties. J Biol Chem 278,
1647–1655.

23 Brade L, Kosma P, Appelmelk BJ, Paulsen H & Brade
H (1987) Use of synthetic antigens to determine the epi-
tope specificities of monoclonal antibodies against the
3-deoxy-d-manno-octulosonate region of bacterial lipo-
polysaccharide. Infect Immun 55, 462–466.
24 Brandenburg K, Richter W, Koch MHJ, Meyer HW &
Seydel U (1998) Characterization of the nonlamellar
cubic and H
II
structures of lipid A from Salmonella
enterica serovar Minnesota by X-ray diffraction and
freeze-fracture electron microscopy. Chem Phys Lipids
91, 53–69.
25 Loppnow H, Libby P, Freudenberg MA, Kraus JH,
Weckesser J & Mayer H (1990) Cytokine induction by
lipopolysaccharide (LPS) corresponds to the lethal toxi-
city and is inhibited by nontoxic Rhodobacter capsulatus
LPS. Infect Immun 58, 3743–3750.
26 Kirikae T, Schade FU, Kirikae F, Qureshi N, Taka-
yama K & Rietschel ET (1994) Diphosphoryl lipid A
derived from the lipopolysaccharide (LPS) of Rhodobac-
ter sphaeroides ATCC 17023 is a potent competitive
LPS inhibitor in murine macrophage-like J774.1 cells.
FEMS Immunol Med Microbiol 9, 237–244.
27 Triantafilou K, Triantafilou M & Dedrick RL (2001) A
CD14-independent LPS receptor cluster. Nature Immu-
nol 2, 338–345.
28 Heine H, Mu
¨
ller-Loennies S, Brade L, Lindner B &

Brade H (2003) Endotoxic activity and chemical struc-
ture of lipopolysaccharides from Chlamydia trachomatis
serotypes E and L2 and Chlamydophila psittaci 6 BC.
Eur J Biochem 270, 440–450.
29 Imoto M, Yoshimura H, Shimamoto T, Sakaguchi N,
Kusumoto S & Shiba T (1987) Total synthesis of
Escherichia coli lipid A, the endotoxically active princi-
ple of cell-surface lipopolysaccharide. Bull Chem Soc
Jpn 60, 2205–2214.
30 Liu W-C, Oikawa M, Fukase K, Suda Y & Kusumoto
S (1999) A divergent synthesis of lipid A and its chemi-
cally stable unnatural analogues. Bull Chem Soc Jpn 72,
1377–1385.
31 Oikawa M, Wada A, Yoshizaki H, Fukase K & Kusu-
moto S (1997) New efficient synthesis of a biosynthetic
precursor of lipid A. Bull Chem Soc Jpn 70, 1435–1440.
32 Imoto M, Yoshimura H, Yamamoto M, Shimamoto T,
Kusumoto K & Shiba T (1987) Chemical synthesis of a
biosynthetic precursor of lipid A. Bull Chem Soc Jpn 60,
2197–2204.
33 Galanos C, Lu
¨
deritz O & Westphal O (1969) A new
method for the extraction of R lipopolysaccharides. Eur
J Biochem 9, 245–249.
34 Koch MHJ & Bordas J (1983) X-ray diffraction and
scattering on disordered systems using synchrotron
radiation. Nucl Instrum Methods
25
208, 461–469.

35 Gabriel A (1977) Position-sensitive X-ray detector. Rev
Sci Instrum 48, 1303–1305.
36 Ju
¨
rgens G, Mu
¨
ller M, Koch MHJ & Brandenburg K
(2001) Interaction of hemoglobin with enterobacterial
lipopolysaccharide and lipid A. Physicochemical charac-
terization and biological activity. Eur J Biochem 268,
4233–4242.
37 Friberger P, So
¨
rskog L, Nilsson K & Kno
¨
s M (1987)
The use of a quantitative assay in endotoxin testing. In
Detection of Bacterial Endotoxin with the Limulus Ame-
bocyte Lysate Test (Watson SW, Levin J & Novitzky
TJ, eds), pp. 149–169. A. Liss, New York.
38 Kuhn HM, Brade L, Appelmelk BJ, Kusumoto S,
Rietschel ET & Brade H (1992) Characterization of the
epitope specificity of murine monoclonal antibodies
directed against lipid A. Infect Immun 60, 2201–2210.
39 Bode CE, Brabetz W & Brade H (1998) Cloning and
characterization of 3-deoxy-d-manno-oct-2-ulosonic acid
(Kdo) transferase genes (kdtA) from Acinetobacter bau-
mannii and Acinetobacter haemolyticus. Eur J Biochem
254, 404–412.
Characterization of carboxymethyl lipid A U. Seydel et al.

340 FEBS Journal 272 (2005) 327–340 ª 2004 FEBS