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
Trang 1Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity
Ulrich Seydel1, Andra B Schromm1, Lore Brade1, Sabine Gronow1, Jo¨rg Andra¨1, Mareike Mu¨ller1, Michel H J Koch2, Koichi Fukase3, Mikayo Kataoka3, Masaya Hashimoto3, Shoichi Kusumoto3 and Klaus Brandenburg1
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
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: useydel@fz-borstel.de
(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 parpar-ticular 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.
Trang 2hydrophobic 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 vitroand 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 reduccomposi-tion of the number of charges or
the number of acyl chains, a change in their
distribu-tion, or degree of saturadistribu-tion, 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,
HII) 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
O
O HO
NH O
O O
OH
O
OH
R 3
R 4
Fig 1 Chemical structures of synthetic tetraacyl and hexaacyl lipid
A analogs P, phosphate groups; R 1 –R 4 , side groups as indicated.
26
Trang 3membrane 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
vs(CH2) for the different compounds reveals a
con-siderable influence of the phosphate substitution in
the case of the tetraacyl compounds The phase
transition temperature Tc of compound 406 around
25C 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 Tc values of the hexaacyl
compounds are very similar and lie slightly above
50C 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 37C (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 [vas(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
2849 2850 2851 2852 2853
2854
406 Bis-CM-406 506 CM-506 Bis-CM-506
–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
vas(COO – ).
Trang 4measured 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–
80C 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
40C, 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 nmÆv5¼ 5.50 nm
and 1.94 nmÆv8¼ 5.50 nm hold At 80 C, the three
observable reflections are clearly indicative of an
inverted hexagonal HII structure, because 4.61 nm¼ 2.67 nmÆv3 and 4.61 nm¼ 2.31 nmÆv4 This phase is already observable at 60C (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 ()
Trang 5polyphemus 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 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 addiinhibi-tion of paxilline can clearly
be observed, in particular at the lower lipid
Bis-CM-406
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.
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 /IDA
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.
Trang 6concentrations, 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
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%.
Trang 7Antagonistic 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)1corresponds to 1 ngÆmL)1of 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
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 paxilpaxil-line
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.
Trang 8Burkholderia cepacia (bifunctional, lane C) [22], and
Chlamydia psittaci
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)
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¨, M Mu¨ller, MHJ Koch, K Fukase, M Kataoka, M Hashimoto,
S Kusumoto & K Brandenburg, unpublished data)
Compound Bis-CM-506 adopts a mixed multilamellar⁄ zcubic aggregate structure at 40C 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
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
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.
Trang 9found 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 vs(CH2) and concomitantly an
increase in Tc (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 capsulatusand Rhodobacter sphaeroides as
well as for lipid A from Chromobacterium violaceum
([5] and U Seydel, AB Schromm, L Brade, S Gronow,
J Andra¨, M Mu¨ller, MHJ Koch, K Fukase, M
Kata-oka, M Hashimoto, S Kusumoto & K Brandenburg,
unpublished data)
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 posisubstitu-tion 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 Bis-CM-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
Trang 10this 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 (Troc¼ 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 OsO4, and the resulting diol was
oxida-tively cleaved with Pb(OAc)4 to give 1-O-formylmethyl
glycoside, which was further oxidized with NaClO2 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 (BF3ÆOEt2
9 and Et3SiH) to give the 6-O-benzyl-4-OH GlcN derivative The benzyloxy carbonyl group was then introduced to the 4-O-position using Ag2O and ICH2
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 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)
phe-nol⁄ chloroform ⁄ petrol ether method [33] from bacteria grown at 37C, 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 C as a 1 mgÆmL)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)