Biophysical characterization of the interaction of
Limulus polyphemus
endotoxin neutralizing protein with lipopolysaccharide
Jo¨ rg Andra¨
1
, Patrick Garidel
2
, Andreja Majerle
3
, Roman Jerala
3
, Richard Ridge
4
, Erik Paus
4
, Tom Novitsky
4
,
Michel H. J. Koch
5
and Klaus Brandenburg
1
1
Forschungszentrum Borstel, Leibniz-Zentrum fu
¨r Medizin und Biowissenschaften, Borstel, Germany;
2
Martin-Luther-Universita
¨t
Halle, Institut fu
¨r Physikalische Chemie, Halle, Germany;
3
National Institute of Chemistry, Laboratory of Biotechnology, Ljubljana,
Slovenia;
4
Associates of Cape Cod, Falmouth, MA, USA;
5
European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o
DESY, Hamburg, Germany
Endotoxin-neutralizing protein (ENP) of the horseshoe crab
is one of the most potent neutralizers of endotoxins [bacterial
lipopolysaccharide (LPS)]. Here, we report on the inter-
action of LPS with recombinant ENP using a variety of
physical and biological techniques. In biological assays
(Limulus amebocyte lysate and tumour necrosis factor-a
induction in human mononuclear cells), ENP causes a
strong reduction of the immunostimulatory ability of LPS
in a dose-dependent manner. Concomitantly, the accessible
negative surface charges of LPS and lipid A (zeta potential)
are neutralized and even converted into positive values. The
gel to liquid crystalline phase transitions of LPS and lipid A
shift to higher temperatures indicative of a rigidification of
the acyl chains, however, the only slight enhancement of the
transition enthalpy indicates that the hydrophobic moiety is
not strongly disturbed. The aggregate structure of lipid A is
converted from a cubic into a multilamellar phase upon ENP
binding, whereas the secondary structure of ENP does not
change due to the interaction with LPS. ENP contains a
hydrophobic binding site to which the dye 1-anilino-8-sulf-
onic acid binds at a K
d
of 19 l
M
, which is displaced by LPS.
Because lipopolysaccharide-binding protein (LBP) is not
able to bind to LPS when ENP and LPS are preincubated,
tight binding of ENP to LPS can be deduced with a K
d
in the
low nonomolar range. Importantly, ENP is able to incor-
porate by itself into target phospholipid liposomes, and is
also able to mediate the intercalation of LPS into the lipo-
somes thus acting as a transport protein in a manner similar
to LBP. Thus, LPS–ENP complexes might enter target
membranes of immunocompetent cells, but are not able to
activate due to the ability of ENP to change LPS aggregates
from an active into an inactive form.
Keywords: endotoxins; IR spectroscopy; LALF; LPS; tumor
necrosis factor.
Lipopolysaccharides (LPS) represent the main amphiphilic
component of the outer leaflet of the outer membrane
of Gram-negative bacteria. They are often referred to as
endotoxins due to their ability to induce a variety of
biological effects in mammals, in particular the production
of proinflammatory cytokines [1]. At low endotoxin
concentrations, the biological effects may be beneficial, as
some cytokines such as tumour necrosis factor-a(TNFa)
have been shown to possess antitumour activity. At higher
endotoxin concentrations, however, the release of cytokines
leads to an inflammatory response, eventually resulting in
the septic shock syndrome [2].
LPS consists of a sugar moiety comprising the O-specific
chain, outer core and inner core covalently linked to the
hydrophobic moiety of LPS, lipid A, which anchors the
LPS molecule to the membrane [2]. The sugar moiety type
and length depends on the strain of bacteria. Smooth strains
have varying length O-specific chains, while rough mutants
have no O-specific chain (Ra) and can also lack parts of the
core regions (Rb–Re). Deep rough mutant LPS (LPS Re)
comprises the inner core, which contains molecules of the
unusual ketose 2-keto-3-deoxyoctonate, each having one
carboxylate group, linked to the lipid A moiety. Lipid A
alone exhibits all of the characteristic endotoxic activities,
and is therefore called the endotoxic principleof LPS [2,3].
In enterobacterial strains, it is composed of a diglucosamine
backbone which is phosphorylated at positions 1 and 4¢and
acylated by 6–7 hydrocarbon chains ester- and amide-linked
in positions 2,3 and 2¢,3¢[3].
LPS of different kinds of bacterial mutants carries a high
number of negative charges. On the basis of these structural
Correspondence to K. Brandenburg, Forschungszentrum Borstel,
Division of Biophysics, Parkallee 10, D-23845 Borstel, Germany.
Fax: + 49 4537 188632, Tel.: + 49 4537 188235,
E-mail: kbranden@fz-borstel.de
Abbreviations: ANS, 1-anilino-8-sulfonic acid; DSC, differential
scanning calorimetry; ENP, endotoxin-neutralizing protein; EU,
endotoxin units; FRET, fluorescence resonance energy transfer; Hb,
hemoglobin; HDL, high-density lipoprotein; LAL, Limulus amebo-
cyte lysate; LALF, Limulus anti-LPS factor; LBP, lipopolysaccharide-
binding protein; LF, lactoferrin; LPS, lipopolysaccharide; LYS,
lysozyme; MNC, mononuclear cells; NBD–PE, N-(7-nitrobenz-2-
oxa-1,3-diazol-4yl)-phosphatidyl ethanolamine; PMB, polymyxin B;
PS, phosphatidylserine; Rh-PE, N-(lissamine rhodamine B sulfonyl)-
phosphatidylethanolamine; rHSA, recombinant human serum
protein; TNFa, tumour necrosis factor-a.
(Received 11 February 2004, revised 25 March 2004,
accepted 31 March 2004)
Eur. J. Biochem. 271, 2037–2046 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04134.x
prerequisites it is therefore not surprising that polycationic
drugs (peptides, proteins, and/or antibiotics) can effectively
protect against the pathophysiological effects of LPS, and
that also the innate immune system provides various
cationic proteins such as lactoferrin, the defensins, the
CAP family [4], and bactericidal permeability-increasing
protein [5], to combat whole bacteria as well as isolated
LPS. One very potent protein with endotoxin-binding and
neutralizing capacity is the Limulus anti-LPS factor (LALF)
from the horseshoe crab Limulus polyphemus [6–8]. This
protein was found to inhibit the endotoxin-mediated
activation of the Limulus coagulation system. LALF is an
11.8-kDa single-chain protein isolated from amebocytes, the
single blood cell type found in these animals [6]. It has been
shown that LALF and its recombinant version, termed
endotoxin-neutralizing protein (ENP), are effective inhibi-
tors of cytokine induction and decreased mortality in
rabbits when administered in vivo before or after LPS
challenge [7]. The tertiary structure of ENP has the same
fold as the C-terminal domain of recA protein [9] which
contains a site capable of binding the hydrophobic probe
1-anilino-8-sulfonic acid (ANS). Reports have been pub-
lished on the protective action against bacteria or LPS of
LALF in in vitro and in vivo systems [7,8,10–14]; however,
no detailed investigation, e.g. into the binding mechanisms,
the target groups, and the understanding of the inhibition
mechanisms has been published so far.
Here, a physiochemical characterization of the ENP/
endotoxin interaction was performed, using enterobacterial
LPS Re and free lipid A. This investigation comprises the
thermodynamics of the lipid/protein interaction, i.e. the
determination of the phase transition behaviour of the acyl
chains of lipid A, its aggregate structure, the accessible
backbone charges, and the binding constant, all at different
ENP concentrations. The protein’s secondary structure at
different endotoxin concentrations and the ability of ENP
to incorporate by itself or to transport LPS into target cell
membranes was also investigated, as was the ability of ENP
to induce change of biological activity of the endotoxins
in the Limulus amebocyte lysate assay and the cytokine
(TNFa) induction in human mononuclear cells. The
data obtained from these measurements allow a model of
LPS–ENP interaction to be established.
Materials and methods
Lipids
LPS from the deep rough mutant Re Salmonella minnesota
(R595) was extracted by the phenol/chloroform/petrol ether
method [15] from bacteria grown at 37 C, purified and
lyophilized. Free lipid A was isolated by acetate buffer
treatment of LPS R595. After isolation, the resulting lipid A
was purified and converted to its triethylamine salt. The
known chemical structure of lipid A from LPS R595 was
checked by the analysis of the amount of glucosamine, total
and organic phosphate, and the distribution of the fatty acid
residues by applying standard procedures. The amount of
2-keto-3-deoxyoctonate never exceeded 5% by weight.
In some cases the LPS S-form from S. minnesota was also
used; this was extracted from bacteria by the phenol/water
procedure [15].
Preparation of endotoxin aggregates
LPS or lipid A was solubilized in the appropriate buffer
(lipid concentration, 1–10 m
M
, depending on the applied
technique), extensively vortexed, sonicated for 30 min, and
subjected to several temperature cycles between 20 and
60 C. Finally, the lipid suspension was incubated at 4 C
for at least 12 h before use.
Proteins
Recombinant ENP expressed in Pichia pastoris, originally
isolated from the American horseshoe crab (Limulus
polyphemus)asLimulus LALF [6,16], was obtained at a
purity > 85% as determined by PAGE and RP-HPLC.
ENP differs from the published native LALF sequence by
four additional N-terminal amino acid residues (EAEA:
Glu-Ala-Glu-Ala) [16].
Lipopolysaccharide-binding protein (LBP) was a kind
gift of S. F. Carroll (XOMA Research, Berkeley, CA, USA).
Determination of endotoxin activity by the chromogenic
Limulus
test
Endotoxin activity of LPS/ENP mixtures 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) [17]. The standard endotoxin
used in this test was from Escherichia coli (O55:B5) and
10 endotoxin units (EU)ÆmL
)1
corresponds to 1 ngÆmL
)1
.
In this assay, saturation occurs at 125 EUÆmL
)1
and the
resolution limit is > 0.1 EUÆmL
)1
(maximum value for
ultrapure, endotoxin-free water, Aqua B. Braun).
Stimulation of human mononuclear cells by LPS
Mononuclear cells (MNC) were isolated from heparinized
blood of healthy donors as described previously [18]. The
experiments were undertaken with the understanding and
written consent of each subject. The cells were resuspended
in medium (RPMI 1640) and their number was equilibrated
at 5 ·10
6
cellsÆmL
)1
. For stimulation, 200 lLMNC
(1 ·10
6
cells)weretransferredintoeachwellofa96-well
culture plate. LPS and LPS/ENP mixtures were incubated
for 30 min at 37 C,andaddedtotheculturesat20lLper
well. The cultures were incubated for 4 h at 37 C under 5%
(v/v) CO
2
. Supernatants were collected after centrifugation
of the culture plates for 10 min at 400 gandstoredat
)20 C until determination of TNFacontent. Immunolo-
gical determination of TNF was carried out in a Sandwich
ELISA using a mAb against TNF (clone 6b from Intex AG,
Switzerland) and has been described earlier in detail [18].
FTIR spectroscopy
The infrared spectroscopic measurements were performed
on an IFS-55 spectrometer (Bruker, Karlsruhe, Germany).
Samples, dissolved in 20 m
M
Hepes buffer pH 7.0, were
placed in a CaF
2
cuvette with a 12.5-lm Teflon spacer.
Measurements were performed at the intrinsic instrument
temperature (26 C), or if indicated, temperature scans were
2038 J. Andra
¨et al. (Eur. J. Biochem. 271)FEBS 2004
performed automatically between 10 and 70 Cwitha
heating rate of 0.6 CÆmin
)1
.Every3C, 50 interferograms
were accumulated, apodized, Fourier transformed, and
converted to absorbance spectra. For strong absorption
bands, the band parameters (peak position, band width,
and intensity) were evaluated from the original spectra, if
necessary after subtraction of the strong water bands. In the
case of overlapping bands, in particular for the analysis of
amide I-vibration mode, curve fitting was applied using a
modified version of the
CURFIT
program obtained from
D. Moffat (NRC, Ottawa, Canada). An estimate of the
number of band components was obtained from deconvo-
lution of the spectra and the curve was fitted to the original
spectra after subtraction of base lines resulting from
neighbouring bands. The bandshapes of the single compo-
nents are superpositions of Gaussian and Lorentzian. Best
fits were obtained by assuming a Gauss fraction of
0.55–0.60. The precision of the curve fit procedure is 3%.
Zeta potential
Zeta potentials were determined with a Zeta-Sizer 4
(Malvern Instr., Herrsching, Germany) at a scattering angle
of 90from the electrophoretic mobility by laser-Doppler
anemometry as described earlier [19]. The Zeta potential
was calculated according to the Helmholtz–Smoluchovski
equation from the mobility of the aggregates in a driving
electric field of 19.2 VÆcm
)1
. Endotoxin aggregates (0.1 m
M
)
and ENP stock solutions (0.5 m
M
)werepreparedin10m
M
Tris, 2 m
M
CsCl pH 7.0.
Differential scanning calorimetry
Differential scanning calorimetry (DSC) measurements were
performed with a MicroCal VP scanning calorimeter
(MicroCal Inc., Northhampton, MA, USA). The heating
and cooling rate was 1 CÆmin
)1
. Heating and cooling curves
were measured in the temperature interval from 10 to
100 C. Usually, three consecutive heating and cooling scans
were measured [20,21]. A stock solution of ENP and an LPS
suspension (both 1 mgÆmL
)1
in phosphate buffer pH 6.8)
were mixed to obtain different ENP/LPS molar ratios.
Fluorescence measurements of binding of ANS to ENP
The fluorescence of the hydrophobic dye ANS was measured
in a luminescence spectrometer LS55 (PerkinElmer) at 25 C
in a quartz cuvette of 1 cm path-length. Excitation wave-
length was 370 nm, and excitation and emission slit widths
5 nm. Fluorescence emission spectra intensities at 470 nm
were determined from titration of 1 l
M
ENP with ANS in
20 m
M
potassium phosphate buffer pH 7.0 after subtraction
of the fluorescence intensities of pure ANS at corresponding
concentrations. The dissociation constant was calculated
from a nonlinear fit of the fluorescence intensity as a function
of ANS concentration using a quadratic equation which
accounts for ligand depletion as described [22,23].
Competitive inhibition of ENP binding to biotinylated LPS
LPS (S-form from S. minnesota) was labelled with biotin
as described previously [24]. Ninetysix-well flat-bottom
microtiter plates (Immuno Plate, MaxiSorp Surface, Nunc)
were coated with ENP (0.5 l
M
)in50lL of coating buffer
(50 m
M
sodium carbonate buffer pH 9.5) at 4 Covernight.
After blocking with 3% (w/v) BSA in NaCl/P
i
for 1 h at
room temperature and washing five times with NaCl/P
i
,
ENP and polymyxin B (PMB) at increasing concentrations
(0, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 l
M
and
0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 and 51.2 l
M
,
respectively) were preincubated with LPS-biotin (3 l
M
)in
50 lLofNaCl/P
i
for 30 min at 37 C. These mixtures were
added to the wells and incubated for additional 2 h at
37 C, followed by washing five times with NaCl/P
i
and
incubation with streptavidin/peroxidase-conjugated poly-
mer (Sigma) diluted 1 : 1000 in NaCl/P
i
for 2 h at 37 C.
Finally, after washing five times with NaCl/P
i
,50lLof
2,2¢-azinobis-3-ethylbenzothiazoline-6-sulfonic acid liquid
substrate (Sigma) was applied to each well and absorbance
at 405 nm was read in a microtiter plate reader 3550-UV
(Bio-Rad). Results were corrected by subtracting absorb-
ance values under the same conditions in the absence of
ENP. The concentration of ENP or PMB that inhibited
50% of binding of immobilized ENP to LPS-biotin was
defined as the I
50
.
X-ray diffraction
X-ray diffraction measurements were performed at the
EMBL outstation at the Hamburg synchrotron radiation
facility HASYLAB using the double-focusing monochro-
mator-mirror camera X33 [25]. Diffraction patterns in the
range of the scattering vector 0.07 < s < 1 nm
)1
(s ¼
2sinh/k,2hscattering angle and kthe wavelength ¼0.15
nm) were recorded at 40 C with exposure times of 2 or
3 min using a linear detector with delay line readout [26].
The s-axis was calibrated with tripalmitate which has a
periodicity of 4.06 nm at room temperature. Details of the
data acquisition and evaluation system can be found
elsewhere [27]. The diffraction patterns were evaluated as
described previously [28] assigning the spacing ratios of the
main scattering maxima to defined three-dimensional
structures. The lamellar and cubic structures are most
relevant here. They are characterized by the following
features: (a) lamellar – the reflections are grouped in
equidistantratios,i.e.1,1/2,1/3,1/4,etc.ofthelamellar
repeat distance dL; (b) cubic – the different space groups of
these nonlamellar three-dimensional structures differ in the
ratio of their spacings. The relationship between reciprocal
spacing s
hkl
¼1/d
hkl
and the lattice constant a is s
hkl
¼
[(h
2
+k
2
+l
2
)/a]
1/2
where hkl are the Miller indices of the
corresponding set of plane.
Fluorescence resonance energy transfer (FRET)
The FRET assay was performed as described earlier [29,30].
Briefly, phospholipid liposomes from phosphatidylserine
(PS) or from a phospholipid mixture corresponding to the
composition of the macrophage membrane [31] were doubly
labelled with the fluorescent dyes N-(7-nitrobenz-2-oxa-1,3-
diazol-4yl)-phosphatidyl ethanolamine (NBD-PE) and
N-(lissamine rhodamine B sulfonyl)-phosphatidylethanol-
amine (Rh-PE) (Molecular Probes, Eugene, OR, USA).
Intercalation of unlabelled molecules into the doubly
FEBS 2004 LPS–ENP interaction (Eur. J. Biochem. 271) 2039
labelled liposomes leads to probe dilution and with that to a
lower FRET efficiency: the emission intensity of the donor
increases and that of the acceptor decreases (for clarity, only
the quotient of the donor and acceptor emission intensity is
shown here).
In all experiments, doubly labelled phospholipid lipo-
somes were prepared and after 50, 100, and 150 s ENP
and LPS were added in different order, each at a final
concentration of 0.01 m
M
. The NBD donor and Rh
acceptor fluorescence intensities were monitored for at least
300 s.
Results
In the following, the influence of ENP on biological action,
physicochemical characteristics of LPS, and the influence of
the latter on the secondary structure of ENP were investi-
gated with a variety of techniques. LPS Re from S. minne-
sota strain R595 was used, and in some cases for better
information on the mechanism of action, the endotoxic
principlelipid A was also used.
Biological endotoxin-neutralizing activity of ENP
The ability of recombinant ENP to inhibit the biological
activity of LPS was tested (a) in the Limulus amebocyte
lysate assay (Limulus clotting cascade) (Fig. 1A), and (b) by
monitoring the TNFaproduction in human mononuclear
cells (MNC) (Fig. 1B). In both assays, almost complete
inhibition was achieved at an ENP/LPS molar ratio of
20 : 1. In the cell test, dose-dependent inhibition can be
observed starting at a molar excess of ENP independent of
the kind of addition of LPS and ENP (common LPS/ENP
mixtures or subsequent addition of pure LPS and ENP with
0.5 h delay). At the two highest ENP/LPS molar ratios
only, the endotoxin-neutralizing activity was lower when
ENP was added 30 min after the addition of LPS (Fig. 1B).
Secondary structure of ENP
The secondary structure of ENP was determined in the
absence and presence of LPS by FTIR from the analysis of
the amide I vibration bands. In the absence of LPS, the
curve-fit analysis of the amide I vibrational band contour
into single band components revealed three major bands
centered around 1673, 1654, and 1628 cm
)1
(Fig. 2A).
These can be assigned to 31% b-turns, 34% a-helical,
and 33% b-sheet structures, and are in accordance with
the crystal structure data for ENP [32].
The addition of LPS had only a negligible influence on
the ENP secondary structure. Even at the highest LPS/ENP
molar ratio tested (1 : 0.6), no significant conformational
changes of the protein except for a small increase in b-turns
occurs (Fig. 2B).
Zeta potential
The zeta potential as an indicator for accessible surface
charges was determined for LPS Re and lipid A in the
presence of increasing amounts of ENP. Pure endotoxins
have a high negative surface charge corresponding to
potential values of )65 to )75 mV, which is increasingly
compensated by the addition of ENP. Complete surface
charge compensation already occurs at a 0.25–0.4 ENP/
endotoxin molar ratio; at higher ENP concentrations
overcompensation was observed (Fig. 3), which is indicative
of the fact that not only pure charge compensation takes
place, but that ENP also binds to other molecular parts of
the endotoxins. In summary, ENP compensates the negative
charges of the endotoxins completely and very efficiently at
rather low ENP/endotoxin ratios as a prerequisite for their
neutralization.
Influence of ENP on the phase transition properties
of endotoxin
Phase behaviour of endotoxins was analysed by DSC (phase
transition temperature and enthalpy) and FTIR (phase
transition behaviour and acyl chain fluidity). DSC meas-
urements were performed in the temperature range
Fig. 1. Influence of ENP on biological activity of LPS. (A) Endotoxin
activity in the chromogenic Limulus amebocyte lysate assay. Two
different LPS Re concentrations (10 ngÆmL
)1
,openbars;
100 ngÆmL
)1
, grey bars) and increasing amounts of ENP were used.
(B) TNFaproduction in human MNC. LPS/ENP mixtures were
incubated with MNC for 4 h at 37 C. The LPS concentration was
10 ngÆmL
)1
. The concentration of TNFareleased into the supernatant
was determined by ELISA (d). In addition, cells were incubated first
with LPS and then with ENP after 30 min at 37 C(n), or vice versa
(h). ENP alone did not induce TNFaproduction.
2040 J. Andra
¨et al. (Eur. J. Biochem. 271)FEBS 2004
10–100 C; however, only the temperature range in which
phase transitions are observed is shown. The gel to liquid
crystalline phase transition of pure LPS Re as detected by
DSC (first heating scan, lowest panel, Fig. 4A) is charac-
terized by an endothermic peak with a maximum at T
c
¼
31 C and a half-width of T
1/2
¼4.5 C. Its phase trans-
ition enthalpy is DH¼38 kJÆmol
)1
. The presence of ENP
at a lipid to protein molar ratio of 1 : 0.1 broadens the
coexistence region of the phase transition considerably
(T
1/2
)6C) and shifts the maximum of the heat capacity
curve to 34 C (Fig. 4A). The observed phase transition
shift implies a gel phase stabilization of the lipid in the
presence of the protein. Two successive heating and cooling
scans of LPS/ENP at a molar ratio of 1 : 0.1 are presented
in Fig. 4B. The first cooling scan represents the recrystal-
lization of the melted hydrocarbon acyl chains from gauche
into all-trans conformations. Basically no hysteresis is
observed. The exothermic heat capacity curve is marked
by a sharp peak at 34.2 C followed by a broader peak. The
baseline of the heat capacity curve is reached below 20 C.
The second heat capacity curve is slightly shifted to higher
Fig. 3. Zeta potential of lipid A and LPS Re. The electrophoretic
mobility of 0.1 m
M
endotoxin aggregates was measured with various
ENP/endotoxin ratios by using laser Doppler anemometry. Lipid A,
s;LPSRe,d.
Fig. 4. DSC of LPS. The differential heat capacity Cp
diff
vs. tem-
perature was determined for various LPS/ENP ratios (A) and for the
LPS/ENP molar ratio of 1 : 0.1 for the first two heating and cooling
scans (B). The LPS concentration was 1 mgÆmL
)1
.
Fig. 2. Secondary structure of ENP. FTIR spectra of ENP (1 m
M
)
are shown in the spectral range of the amide I vibrational band
(1700–1600 cm
)1
) in the absence (A) and (B) presence of LPS Re
(LPS/ENP ¼1 : 0.6 molar). The band component close to 1650 cm
)1
is indicative of a-helical, that at 1628 cm
)1
of b-sheets, and that at
1673 cm
)1
of b-turns.
FEBS 2004 LPS–ENP interaction (Eur. J. Biochem. 271) 2041