HDL exhibited an increase in the gel to liquid crystalline phase transition temperature Tcand a rigidification of the acyl chains of the endotoxins as measured by Fourier-transform infrar
Trang 1Biophysical characterization of the interaction of high-density
lipoprotein (HDL) with endotoxins
Klaus Brandenburg1, Gudrun Ju¨rgens1, Jo¨rg Andra¨1, Buko Lindner1, Michel H J Koch2, Alfred Blume3 and Patrick Garidel3
1
Forschungszentrum Borstel, Biophysik, Borstel, Germany;2European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Hamburg, Germany;3Martin-Luther-Universita¨t Halle/Wittenberg, Institut fu¨r Physikalische Chemie, Halle, Germany
The interaction of bacterial endotoxins [lipopolysaccharide
(LPS) and the endotoxic principle lipid A], with
high-den-sity lipoprotein (HDL) from serum was investigated with a
variety of physical techniques and biological assays HDL
exhibited an increase in the gel to liquid crystalline phase
transition temperature Tcand a rigidification of the acyl
chains of the endotoxins as measured by Fourier-transform
infrared spectroscopy and differential scanningcalorimetry
The functional groups of the endotoxins interacting with
HDL are the phosphates and the diglucosamine backbone
The findingof phosphates as target groups is in accordance
to measurements of the electrophoretic mobility showing
that the zeta potential decreases from)50 to )60 mV to
)20 mV at bindingsaturation The importance of the sugar
backbone as further target structure is in accordance with the
remainingnegative potential and competition experiments
with polymyxin B (PMB) and phase transition data of the
system PMB/dephosphorylated LPS Furthermore,
endo-toxin bindingto HDL influences the secondary structure of
the latter manifestingin a change from a mixed a-helical/ b-sheet structure to a predominantly a-helical structure The aggregate structure of the lipid A moiety of the endotoxins as determined by small-angle X-ray scattering shows a change
of a unilamellar/inverted cubic into a multilamellar structure
in the presence of HDL Fluorescence resonance energy transfer data indicate an intercalation of pure HDL, and of [LPS]–[HDL] complexes into phospholipid liposomes Fur-thermore, HDL may enhance the lipopolysaccharide-bind-ingprotein-induced intercalation of LPS into phospholipid liposomes Parallel to these observations, the LPS-induced cytokine production of human mononuclear cells and the reactivity in the Limulus test are strongly reduced by the addition of HDL These data allow to develop a model of the [endotoxin]/[HDL] interaction
Keywords: endotoxin conformation; high density lipopro-teins (HDL); lipopolysaccharides; Fourier-transform infra-red spectroscopy
Bacterial lipopolysaccharides (LPS) belongto the most
potent stimulators of the immune system and play an
important role in the pathogenesis and manifestation of
Gram-negative infections, in general, and of septic shock,
in particular, and are thus called endotoxins The
mechanism of endotoxin interaction with different target
cell structures are still largely unknown and only limited
data are available on the detailed mode of bindingof
endotoxins to various endogenous proteins, which are
important with regard to combat invading
microorgan-isms and to transport and neutralize free endotoxin
Amongthe humoral factors which are important
LPS-binding molecules are serum lipoproteins It was
sugges-ted that sequesteringof LPS by lipid particles may form
an integral part of humoral detoxification [1] Lipo-proteins are water-soluble complexes with a neutral core, surrounded by a phospholipid layer that contains cholesterol and one or more apolipoproteins They serve
as ligands for cell membrane receptors, as cofactors for enzymes, and can dock lipopolysaccharide-bindingpro-teins They are classified as very-low density, low-density and high-density lipoproteins (HDL) according to their buoyant density The primary function of these lipo-proteins is to transport lipids, cholesterol and cholesteryl esters in blood and the lymphatic system HDL moreover plays a role in bindingand neutralizingbacterial lipopolysaccharide and decrease the immunostimulatory action of LPS In particular, a drastic reduction of the LPS-induced cytokine production [tumor necrosis
factor-a, interleukin (IL)-1, IL-6] due to HDL bindingwas observed [2–4] Furthermore, it was demonstrated that lipopolysaccharide-bindingprotein (LBP) increased the uptake of LPS by reconstituted HDL (R-HDL) particles derived from either LPS micelles or LPS–sCD14 com-plexes, and in this process LPS molecules are exchanged with phospholipids [5]
Here, we report on the interaction of HDL with deep rough mutant LPS Re and the endotoxic principle, lipid
A applyinga variety of physical and biological techniques With Fourier-transform infrared spectroscopy (FTIR) the phase transition behavior of the acyl chains of the
Correspondence to K Brandenburg, Forschungszentrum Borstel,
Biophysik, Parkallee 10, D-23845 Borstel, Germany.
Fax: +49 4537 188632, Tel.: + 49 4537 188235,
E-mail: kbranden@fz-borstel.de
Abbreviations: ATR, attenuated total reflectance; FTIR,
Fourier-transform infrared spectroscopy; HDL, high-density lipoprotein;
IL, interleukin; LAL, Limulus amebocyte lysate; LBP,
lipo-polysaccharide-bindingprotein; LPS, lipopolysaccharide; PMB,
polymyxin B; PtdSer, phosphatidylserine.
(Received 2 September 2002, revised 18 October 2002,
accepted 24 October 2002)
Trang 2endotoxins in absence and presence of HDL as well as
the effect of HDL on functional groups of the endotoxins
were observed for the latter, usingthe attenuated total
reflectance (ATR) method To obtain information about
the phase transition enthalpy changes of the endotoxins,
differential scanningcalorimetry in the absence and
presence of HDL was carried out Also, with FTIR the
influence of endotoxin bindingon the secondary structure
of the protein part of HDL, apolipoprotein A-I (apoA-I)
was observed The effect of HDL on the surface charge of
the endotoxin aggregates was studied by applying zeta
potential measurements, which also enabled an estimate
for the binding saturation to be made The aggregate
structure and, with that, the conformation of the lipid A
part of LPS was studied by small-angle X-ray diffraction
With fluorescence resonance energy transfer experiments,
information about the influence of HDL on the
inter-calation of LPS and LBP, and the interinter-calation of the
lipoprotein itself into phospholipid target membranes
could be given Finally, in biological experiments the
ability of the endotoxin and [endotoxin]/[HDL] complexes
to induce cytokine production in mononuclear cells and to
activate the Limulus amebocyte lysate (LAL) clotting
cascade was measured Thus, it was possible to
charac-terize the bindingof HDL to the endotoxins profoundly
and to get insight into the mechanisms of the reduction of
the LPS-induced cytokine production in human
mono-nuclear cells
M A T E R I A L S A N D M E T H O D S
Lipids and reagents
Lipopolysaccharide from the deep rough mutant Re
Salmonella minnesota(R595) was extracted by the phenol/
chloroform/petrol ether method [6] from bacteria grown at
37C, purified, and lyophilized Free lipid A was isolated by
acetate buffer treatment of LPS R595 After isolation, the
resultinglipid A was purified and converted to its
triethyl-amine 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 applyingstandard procedures The amount
of 2-keto-3-deoxyoctonate never exceeded 5 weight %
Dephospho-LPS Re was prepared from LPS deep rough
mutant F515 from Escherichia coli by HF treatment at low
temperature (4C) The detailed procedure is described
elsewhere [7]
High-density lipoprotein (HDL) from human plasma was
purchased from Fluka (Deisenhofen, Germany) It was
essentially free of contaminants, in particular of LPS, which
was examined by applyingthe Limulus test (see later)
Lipopolysaccharide-bindingprotein (LBP) was a kind
gift of S F Carroll (XOMA corporation, Berkeley, CA,
USA)
Sample preparation
The lipid samples were usually prepared as aqueous
disper-sions at high buffer content, i.e above 60% using 20 mM
Hepes (pH 7) For this, the lipids were suspended directly in
buffer, sonicated and temperature-cycled several times
between 5 and 70C and then stored for at least 12 h before measurement For the elucidation of the protein secondary structure in the absence and presence of endo-toxins, HDL was prepared in buffer made either from H2O
or D2O incubated at 37C for 30 min, and lipid dispersions prepared as described above were added in appropriate amounts, and further incubated at 37C for 15 min Afterwards, 10 lL of these dispersions were spread on a CaF2infrared window, and the excess water was evaporated slowly at 37C
FTIR spectroscopy The infrared spectroscopic measurements were performed
on a 5-DX FTIR spectrometer (Nicolet Instruments, Madison, WI, USA) and on an IFS-55 spectrometer (Bruker, Karlsruhe, Germany) The lipid samples were placed in a CaF2 cuvette with a 12.5-lm Teflon spacer Temperature-scans were performed automatically between
10 and 70C with a heating-rate of 0.6 CÆmin)1 Every
3C, 50 interferograms were accumulated, apodized, Fou-rier transformed and converted to absorbance spectra For strongabsorption bands, the band parameters (peak position, band width, and intensity) were evaluated from the original spectra, if necessary after subtraction of the strongwater bands
In the case of overlappingbands, in particular for the analysis of amide I-vibration mode, curve fittingwas applied usinga modified version of the CURFIT program obtained by D Moffat, NRC, Ottawa, Canada An estimate of the number of band components was obtained from deconvolution of the spectra [8] and the curve was fitted to the original spectra after subtraction of base lines resultingfrom neighboringbands The bandshapes of the single components are superpositions of Gaussian and Lorentzian Best fits were obtained by assuminga Gauss fraction of 0.55–0.60 The precision of the curve fit procedure is approximately 3%
ATR The lipids were prepared as oriented thin multilayers as described previously [9] by spreadinga 1-mMlipid suspen-sion, which was temperature-cycled between 5 and 70C several times prior to spreading, in Hepes buffer on a ZnSe ATR crystal and evaporatingthe 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 saturated with water vapor to maintain full hydration Infrared ATR spectra were recorded with a mercury–cadmium–telluride detector with a scan number of 1000 at a resolution of
2 cm)1 The measurements were performed at 26C, the intrinsic instrument temperature, in some cases also at
37C
Differential scanning calorimetry LPS was dispersed in buffer at a concentration of
1 mgÆmL)1 A liposomal lipid dispersion was obtained by sonication for 10 min at 40C After coolingto room temperature, a defined amount of HDL was added to 1 mL lipid dispersion and the sample was gently vortexed until
Trang 3HDL was completely dissolved [9] Differential scanning
calorimetry measurements were performed with a MicroCal
VP scanningcalorimeter (MicroCal, Inc., Northampton,
MA, USA) The heatingand coolingrate was 1CÆmin)1
Heatingand coolingcurves were measured in the
tempera-ture interval from 10 to 100C Three consecutive heating
and coolingscans were measured [10]
X-ray diffraction
X-ray diffraction measurements were performed at the
European Molecular Biology Laboratory (EMBL)
outsta-tion at the Hamburgsynchrotron radiaoutsta-tion facility
HASY-LAB usingthe double-focusingmonochromator-mirror
camera X33 [11] Diffraction patterns in the range of the
scatteringvector 0.07 < s < 1 nm)1(s¼ 2 sin hÆk)1, 2h
scatteringangle and k the wavelength¼ 0.15 nm) were
recorded at 40C with exposure times of 2 or 3 min usinga
linear detector with delay line readout [12] The s-axis was
calibrated with tripalmitate, which has a periodicity of
4.06 nm at room temperature Details of the data
acquisi-tion and evaluaacquisi-tion system can be found elsewhere [13] The
diffraction patterns were evaluated as described previously
[14] assigningthe spacingratios of the main scattering
maxima to defined 3D structures The lamellar and cubic
structures are most relevant here They are characterized by
the followingfeatures: (a) lamellar: The reflections are
grouped in equidistant ratios, i.e 1, 1/2, 1/3, 1/4, etc of the
lamellar repeat distance dL; (b) cubic: The different space
groups of these nonlamellar 3D structures differ in the
ratio of their spacing The relation between reciprocal
spacing shkl¼ 1/dhkl and lattice constant a is shkl¼
[(h2+ k2+ l2)/a]1/2, where hkl are Miller indices of the
correspondingset of plane
Zeta potential
Zeta potentials were determined with a Zeta-Sizer 4
(Malvern Instr., Herrsching, Germany) at a scattering angle
of 90 from the electrophoretic mobility by laser-Doppler
anemometry as described earlier [15] The zeta potential was
calculated accordingto the Helmholtz-Smoluchovski
equa-tion from the mobility of the aggregates in a driving electric
field of 19.2 VÆcm)1 It was determined for the endotoxins
(0.5 mM) at different HDL concentrations
Isothermal titration calorimetry
Microcalorimetric experiments of HDL-bindingto
endo-toxins were performed on an MCS isothermal titration
calorimeter (Microcal Inc., Northampton, MA, USA) The
endotoxin samples at a concentration of 0.25 mgÆmL)1,
prepared as described above, were filled into the
microca-lorimetric cell (volume 1.3 mL), and HDL at concentrations
up to 12 mgÆmL)1were loaded into the syringe
compart-ment, both after thorough degassing of the suspensions
After temperature equilibration, the HDL was titrated in
5 lL portions every 10 min into the endotoxin-containing
cell, and the heat for each injection measured by the ITC
instrument was plotted vs time The total heat signal from
each experiment was subsequently determined by
integra-tingthe individual peaks and plotted against the [HDL]/
[endotoxin] weight ratio
Fluorescence resonance energy transfer The fluorescence resonance energy transfer assay was per-formed as described earlier [16,17] Briefly, phospholipid liposomes from phosphatidylserine (PtdSer) were doubly labeled with the fluorescent dyes N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)-phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rh-PE) (Molecular Probes, Eugene, OR, USA) Intercalation of unlabeled molecules into the doubly labeled liposomes leads
to probe dilution and thus inducinga lower fluorescence resonance energy transfer 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 labeled PtdSer liposomes were prepared and after 50, 100, and 150 s recombinant LBP, LPS, and HDL were added in different order, and the NBD donor fluorescence intensity at 531 nm was monitored for at least 300 s LBP, HDL and LPS were added in the weight ratios 0.5 : 1 : 1
Stimulation of human mononuclear cells by LPS Re For an examination of the cytokine-inducingcapacity of the [endotoxin]/[HDL] mixtures, human mononuclear cells were stimulated with the latter and the IL-6 production of the cells was determined in the supernatant
Mononuclear cells were isolated from heparinized (20 IEÆmL)1) blood taken from healthy donors and processed directly by mixingwith an equal volume of Hank’s balanced solution and centrifugation on a Ficoll density gradient for
40 min (21C, 500 g) The layer of mononuclear cells was collected and washed twice in Hank’s medium and once in serum-free RPMI 1640 containing2 mM L-glutamine,
100 UÆmL)1penicillin, and 100 lgÆmL)1streptomycin The cells were resuspended in serum-free medium and their number was equilibrated at 5· 106cellsÆmL)1 For stimu-lation, 200 lLÆwell)1mononuclear cells (5· 106cellsÆmL)1) were transferred into 96-well culture plates The stimuli were serially diluted in serum-free RPMI 1640 and added to the cultures at 20 lL per well The cultures were incubated for 4
h at 37C under 5% CO2 Supernatants were collected after centrifugation of the culture plates for 10 min at 400 g and stored at)20 C until determination of cytokine content Immunological determination of IL-6 in the cell super-natant was performed in a sandwich-ELISA as described elsewhere [18] Ninety-six-well plates (Greiner, Solingen, Germany) were coated with a monoclonal (mouse) human IL-6 antibody (clone 16 from Intex AG, Switzerland) Cell culture supernatants and the standard (recombinant human IL-6, Intex) were diluted with buffer After exposure to appropriately diluted test samples and serial dilutions of standard rIL-6, the plates were exposed to peroxidase-conjugated (sheep) anti-human IL-6 antibody The plates were shaken 16–24 h at room temperature (21–24C) and washed six times in distilled water to remove the antibodies Subsequently the color reaction was started by addition
of tetramethylbenzidine/H2O2 in alcoholic solution and stopped after 5–15 min by addition of 0.5 molÆL)1sulfuric acid In the color reaction, the substrate is cleaved enzymatically, and the product was measured photometri-cally on an reader (Rainbow, Tecan, Crailsham,
Trang 4Germany) at a wavelength of 450 nm and the values were
related to the standard IL-6 was determined in duplicate at
two different dilutions and the values were averaged
Determination of endotoxin activity by the
chromogenicLimulus test
Endotoxin activity of [LPS]–[HDL] mixtures at
concentra-tions between 10 lgÆmL)1and 10 pgÆmL)1was determined
by a quantitative kinetic assay based on the reactivity of
Gram-negative endotoxin with LAL [19], using test kits
from LAL Coamatic Chromo-LAL K (Chromogenix,
Haemochrom) The standard endotoxin used in this test
was from E coli (O55:B5), and 10 EUÆmL)1corresponds to
1 ngÆmL)1 In this assay, saturation occurs at 125 endotoxin
units EUÆmL)1, and the resolution limit is £ 0.1 EUÆmL)1
(maximum value for ultrapure water from embryo-transfer,
Sigma)
R E S U L T S
Measurements of hydrated LPS–HDL complexes
Infrared-ATR experiments were performed with hydrated
LPS multilayers in the absence and presence of different
HDL concentrations In these measurements, the LPS
concentration was held constant and the spectra were
normalized by takingthe band intensity of the symmetric
stretchingvibration ms(CH2) as standard In Fig 1, a change
in the band contours in the range of the two phosphate and
the diglucosamine vibrations, mas(PO2) 1270–1250 cm)1,
and mas(PO2)hydr. 1230–1220 cm)1 and mas(diglucosamine)
1180–1150 cm)1, can be seen; the addition of HDL leads to
an intensity decrease in the band contours proportional to
the HDL concentration From Fig 1 it can be taken that
especially the intensity of the band component around
1190 cm)1 increases as compared with that at lower
wavenumbers and becomes sharper Additionally, the
component at 1170 cm)1 for pure LPS is shifted to approximately 1177 cm)1in the presence of HDL These results indicate that (i) besides the phosphate groups, the sugar diglucosamine part in lipid A are also binding-sites for HDL, and (ii) these vibrational bands are immobilized due
to HDL binding
Gel to liquid crystalline (b«a) phase behavior The b«a gel to liquid crystalline acyl chain melting behavior was investigated with FTIR by evaluating the peak position
of the symmetric stretchingvibration ms(CH2), which is a measure of acyl chain order HDL induces a slight rigidification in particular in the liquid crystalline (a) phase
of the acyl chains of LPS Re, as deduced from a decrease in wavenumber values at a given temperature, and a significant increase in the phase transition Tcfrom 31C for pure LPS
to 40C for an [LPS]–[HDL] mixture at a weight ratio of
1 : 4 Also, pure HDL exhibits a signal in this wavenumber range due to its phospholipid moiety This, however, is much higher with an only weak temperature dependence in the wavenumber range 2852.5–2853.5 cm)1(Fig 2) These values are indicative of acyl chains with a large amount of gauche conformers Importantly, the interaction of HDL with LPS leads to a reduction of the wavenumber by more than one unit (see vertical line at 37C), i.e a strong rigidification of the lipid A acyl chains
This holds true also for lipid A even although higher amounts of HDL are required to induce a significant increase in Tc Thus, at a weight ratio[lipid A]/[HDL] 1 : 3 the phase transition at Tc¼ 45 C of pure lipid A is shifted
to 50C (data not shown) This observation reflects the different number of negative charges and monosaccharide units (LPS Re has four negative charges and four sugar units, lipid A two of each) which may be connected with different conformations of the molecules
Differential scanningcalorimetry measurements of the interaction of LPS with HDL (Fig 3) shows for pure LPS a phase transition in accordance to that observed in Fig 2
Fig 1 Infrared-ATR spectra in the range of the antisymmetric
stretching vibration of the negatively charged phosphate groups
m as (PO 2 )1210–1260 cm)1) and the diglucosamine ring vibration (see
arrows) of LPS at different [LPS]/[HDL] weight ratios The spectra
were normalized by takingthe band intensity of the symmetric
stretchingvibration of the methylene groups m (CH ) as standard.
Fig 2 Peak position of the symmetric stretching vibration of the methylene groups m s (CH 2 ) vs temperature for a 10-m M LPS Re pre-paration at different HDL concentrations In the gel (b) phase of the acyl chains, the peak position lies at 2850 cm)1, in the liquid crystalline (a) phase at 2852.5 cm)1.
Trang 5The phase transition in the first heatingscan is characterized
by a coexistence region between 22 and 37C (T1/2¼
4.5C) and the maximum of the heat capacity curve is
found at 31C with DHC¼ 38 kJÆmol)1 The succeeding
coolingscan reveals only a very small hysteresis for the
re-crystallization of the acyl chains from the liquid
crystal-line to the gel phase The maximum of the heat capacity
curve of the 1st coolingscan is observed at T¼ 28 C with
DH¼)39 kJÆmol)1 A shoulder at 23 C is observed in
the first and succeedingcoolingscan The thermograms of
the succeedingheatingscan are slightly broader compared
with the 1st heatingscan (Fig 3A)
HDL was added to LPS at different concentrations
{[LPS]/[HDL] 1 : 0.25, 1 : 0.45, 1 : 0.6 and 1 : 1 (w/w)} In
Fig 3(B) representative thermograms for the sample at a
LPS/HDL 1 : 1 (w/w) ratio are plotted The phase
trans-ition temperature of LPS is shifted from 31C to 33 C,
the half-width of the phase transition is increased
(T1/2¼ 7 C) and the phase transition enthalpy is decreased
by 22% The presence of HDL induces a broadeningof
the coexistence range of the phase transition, especially for
the offset temperature which is shifted above 42C The
phase transition as derived from the IR spectra from the
temperature dependence of ms(CH2) of the [LPS]/[HDL]
1 : 0.5 system revealed similar data: Tc¼ 34 C and
T1/2¼ 8.5 C The heat-capacity curve of LPS/HDL ratio
develops a shoulder startingat 20 C in the gel phase
indicatingthat HDL interacts with the gel phase LPS This
is observed for all four investigated LPS/HDL
concentra-tion ratios A second peak with a very small enthalpy
contribution at higher temperature (T 63 C, DH ¼
8 kJÆmol)1) corresponds to the denaturation peak of
pure HDL, because the maximum of the heat capacity curve
of pure HDL is observed at 63 C (Fig 3C) Thus,
additional HDL does not interact with the LPS membrane
but acts like pure protein Heatingof the sample above
70 C leads to complete and irreversible denaturation of
HDL (data not shown)
Parallel to the measurements of LPS Re, differential
scanningcalorimetry measurements of the phase behavior
of lipid A indicated a similar increase in Tc, and the
evaluation of the phase transition enthalpy (peak area)
showed a value of 14 kJÆmol)1 which in the presence of
HDL is reduced to 12 kJÆmol)1, i.e a reduction by 15% These data indicate that the bindingof HDL to LPS and lipid A leads to a disturbance of the hydrophobic moiety Inhibition experiments were performed with the polycat-ionic peptide polymyxin B (PMB), which binds strongly to the lipid A phosphates [20] At a [LPS]/[PMB] weight ratio
of 1 : 0.24, PMB alone causes a drastic fluidization of LPS, while HDL leads to a rigidification of LPS at a weight ratio
of [LPS]/[HDL] 1 : 1.5 (Fig 4) Addition of HDL to the preincubated [LPS]–[PMB] complex leads to almost the same result as without HDL, and addition of PMB to preincubated [LPS]–[HDL] causes a slightly attenuated fluidizingeffect as compared with LPS with PMB alone PMB, which binds much stronger to the LPS phosphates than HDL, may displace HDL molecules from their binding site, the lipid A phosphates
These results are complemented by the data of the dephospho-LPS Re and HDL systems (Fig 5) Dephos-pho-LPS Re has a Tc of 45C, and in the case of phosphates as the primary bindingsite no change of the phase behavior of dephospho-LPS Re would be expected However, addition of HDL causes a fluidization parti-cularly in the gel phase and in the transition range at a
Fig 3 Differential scanning calorimetry heat capacity curves of pure LPS Re (A), a mixture
of [LPS]/[HDL] at 1.1 : 1 w/w (B), and for pure HDL (C) Heatingand coolingcurves were measured in the temperature interval 10–
100 C Three consecutive heatingand cooling scans are presented (A,B) (h.s heating-scan, c.s coolingscan) and first heatingscan (C).
Fig 4 Peak position of the symmetric stretching vibration of the methylene groups m s (CH 2 ) vs temperature in competition experiments with LPS Re, PMB and HDL in different sequences.
Trang 6weight ratio [dephospho-LPS]/[HDL] 1 : 4.5 As a control,
the effect of PMB on dephospho-LPS Re was monitored It
is found that PMB causes a slight decrease in Tc, but no
change in fluidity takes place (data not shown) Therefore,
the phosphates can be assumed not to be the only
binding-sites for HDL
LPS and lipid A aggregate structures
Synchrotron radiation X-ray small-angle diffraction was
performed with lipid A at 40C and at different
concentrations of HDL The diffraction patterns of pure
lipid A (Fig 6, top) are indicative of a superposition of a
unilamellar with a cubic inverted structure in accordance
to former results [21], which can be deduced from the
occurrence of the broad interference maximum between
0.1 and 0.4 nm)1 superimposed by diffraction maxima
at 8.20 nm¼ 18.4 nmÆ5, 5.31 nm¼ 18.4 nmÆ12,
4.08 nm¼ 18.4 nmÆ20 of a periodicity at aQ¼
(18.3 ± 0.3) nm (the latter is expressed only very
weakly) In the presence of HDL, this mixed structure
converts into a multilamellar one, which can be deduced
from the occurrence of reflections at equidistant ratios,
d|¼ 5.13 nm and 2.60 nm ¼ d|/2 and 1.74 nm¼ d|/3
(Fig 6, bottom) From these data an approximation of
the molecular shape of lipid A is possible: In the absence
of HDL, it is conical with a higher cross-section of the
hydrophobic than the hydrophilic moiety, and is
conver-ted into a cylindrical one in the presence of HDL
HDL secondary structure
The secondary structure of the apolipoprotein (apoA-I)
part of HDL was determined by IR-spectroscopy by
analyzingthe amide I-vibration (predominantly C¼O
stretchingvibration) in the spectral range 1700–1600 cm)1
in H2O-containingas well as D2O-containingbuffer IR
spectra are given in the range 1700–1400 cm)1at a [LPS]/
[HDL] ratio of 1 : 0.5 weight percentage in D2O (Fig 7A)
exhibitingthe amide I¢-vibration centered around
1653 cm)1, but only a very weak amide II-vibration due
to H/D exchange [22] The evaluation of the amide I¢
vibrational band shows that for HDL in the presence of
Re-LPS ([HDL]/[Re-LPS] 1 : 0.5 weight ratio) in Fig 7B the b-turn/antiparallel b-sheet components of the protein’s secondary structure is changed in favor of the a-helical component (a detailed assignment of the different secon-dary structures is presented in the legend of Fig 7) For pure HDL the a-helical portion is approximately 34% and for the complexes ([HDL]/[Re-LPS] 1 : 0.5 weight ratio) approximately 44% From the broadeningof the
1653 cm)1 band it becomes obvious that a more hetero-geneous population of a-helical structures emerge as consequence of bindingto LPS The occurrence of unordered (random coil) structures, which in D2O are located in the range 1640–1645 cm)1, can be excluded, as measurements in H2O, for which the unordered structures are found around 1660 cm)1, exhibited a similar band contour except for the fact that the peak position of the amide I vibration is shifted to approximately 1658 cm)1 Zeta potential
The zeta potential as an indicator for accessible surface charges was determined for LPS Re and lipid A in the presence of increasingamounts of HDL From Fig 8 it can
be deduced that the pure endotoxins have a high negative surface charge corresponding to potential values of)50 to
Fig 5 Peak position of the symmetric stretching vibration of the
methylene groups m s (CH 2 ) vs temperature for a 10-m M dephospho-LPS
Re preparation at different HDL concentrations.
Fig 6 Synchrotron radiation X-ray diffraction patterns of lipid A (top) and a mixture of lipid A and HDL (bottom, weight ratio 1 : 0.5) at 90% water content The diffraction pattern of the aggregate structure of lipid
A indicates the existence of a superposition of a unilamellar with a cubic inverted structure, that of the mixture a multilamellar structure.
Trang 7)60 mV, which is increasingly compensated by the addition
of higher amounts of HDL However, the charge
compen-sation seems to be completed at a weight ratio [endotoxin]/
[HDL] 1 : 1 at a remainingpotential of)20 mV Therefore,
HDL does not compensate the negative charges of the
endotoxins completely
Isothermal titration calorimetry
With ITC an estimate of the stoichiometry of HDL–LPS
bindingcan be obtained For this, a LPS dispersion
(0.25 mgÆmL)1) within the calorimeter cell was titrated with
a HDL solution (5 lL of 12 mgÆmL)1every 10 min) The
titration yields a negative enthalpy change DHcof the LPS–
HDL bindingcorrespondingto an exothermic reaction
(data not shown) A maximum of DHc¼)14 kJ is
observed at a weight ratio of 1 : 1 At higher [HDL]
contents, the DHcvalues decrease to)6 kJ at weight ratios
[HDL]/[LPS]¼ 4 : 1–6 : 1, but do not decrease to zero
Unfortunately, the HDL amounts available did not allow to
realize higher HDL concentrations, i.e to determine the
saturation of binding, which therefore must be significantly
higher than a weight ratio of [HDL]/[LPS]¼ 6 : 1
Intercalation into phospholipid liposomes
It has been shown that LBP mediates the transport of LPS into negatively charged liposomes [17] which seems to be an important step in cell activation Here, the LBP-mediated transport of LPS into PtdSer as example of negatively charged phospholipids was determined by fluorescence resonance energy transfer spectroscopy in the absence and presence of HDL (Fig 9) The addition of LPS at t¼ 50 s indicates that LPS itself does not intercalate into the PtdSer liposomes, the followingaddition of LBP at t¼ 150 s leads
to an rapid increase in NBD-fluorescence intensity corres-pondingto the LBP-mediated intercalation of LPS and LBP into the PtdSer liposomes (Fig 9A) The addition of HDL
at t¼ 50 s leads to an increase in the NBD-fluorescence intensity indicatingan intercalation of HDL into the PtdSer liposomes, the subsequent addition of LPS at t¼ 100 s apparently leads to an HDL-mediated transport of LPS into the target cell membrane (Fig 9B), as the addition of pure buffer instead of LPS at this time causes a reduction of the fluorescence intensity due to dilution (data not shown) The final addition of LBP at t¼ 150 s leads to another increase in the NBD-fluorescence intensity caused by intercalation of pure LBP and LBP-mediated intercalation
of LPS into the PtdSer liposomes (Fig 9B) In Fig 9C, the addition of LPS first and then of HDL again showed no intercalation of LPS by itself, an intercalation of HDL as found already in Fig 9(B), and the final strong increase of the NBD-fluorescence intensity indicates the intercalation of LBP and the [LPS]–[LBP] complex In Fig 9D, after addition of the preincubated complex (LPS + HDL) the increase of the NBD-fluorescence intensity indicates an intercalation of HDL and (LPS + HDL) complex, which
is followed by the strongincrease due to LBP-induced intercalation
Similar results are obtained when the PtdSer is replaced
by phospholipid liposomes correspondingto the composi-tion of the macrophage membrane [16], only the effects are significantly weaker
IL-6-production in mononuclear cells IL-6 production in human mononuclear cells induced
by LPS Re (10 ngÆmL)1) was investigated at different HDL concentrations The concentration of LPS Re was
Fig 7 Infrared spectra in the range 1700–1400 cm)1for a hydrated sample of [LPS]/[HDL] = 1 : 2 weight ratio (A) and in the range of the amide I¢ (predominantly C=O stretch) vibration for hydrated samples of HDL (B, top) and in the presence of Re-LPS ([LPS]/[HDL] = 1 : 2 weight ratio) (B, bottom) The measurements were performed at 37 C in D 2 O Band component assignments: 1653 cm)1, a-helix; 1636–1638 cm)1, b-sheet; 1667–
1671 cm)1, b-turns; 1682–1685 cm)1, b turns and antiparallel b sheet In the figures, the values of the peak positions and the respective bandwidths (determined at half heig ht, in cm)1) are listed The curve fittingwas performed by assuminga Gaussian fraction of 0.6 (Lorentzian fraction 0.4) The precision of the secondary structural determination is approximately 3%, obtained from repeated measurements (n ¼ 5).
Fig 8 Zeta potential of 0.5 m M lipid A and LPS Re preparations
in dependence on different [endotoxin]/[HDL] weight ratios from the
determination of the electrophoretic mobility by laser Doppler
anemometry.
Trang 8held constant (10 ngÆmL)1) while the HDL concentration
was increased (10 ngÆmL)1, 100 ngÆmL)1, 1 lgÆmL)1,
10 lgÆmL)1) to produce the weight ratios shown As plotted
in Fig 10, three types of experiments with LPS Re and
HDL have been carried out Preincubation of the cells
with LPS Re (30 min at 37C) and followingaddition of
HDL, preincubation of the cells with HDL and following
addition of LPS Re, and the incubation of the cells with
(LPS + HDL) complexes Preincubation of the cells with
HDL and followingaddition of LPS Re, and the (LPS +
HDL) complex leads in all examined concentrations to a
decrease in IL-6 production Preincubation of the cells with
LPS Re and followingaddition of HDL leads to an
insignificant increase in IL-6 production at the lowest HDL
concentration ([LPS]/[HDL] 1 : 1 weight ratio), but at higher concentrations of HDL also to a decrease in the IL-6 production, but less as compared with the results in the other experiments
Biological activity in the LAL assay The ability of the LPS–HDL complexes to activate the LAL clottingcascade was measured at LPS Re concentrations of
100 pgÆmL)1and 1 ngÆmL)1 As shown in Fig 11 we have found that HDL reduces the enzymatic activity induced by pure LPS Re at all investigated concentrations For example, at 1 ngÆmL)1the activity of 45 EUÆmL)1for the pure LPS Re is reduced to values in the range 15–25 EUÆmL)1at all concentrations, startingfrom [LPS]/[HDL]
1 : 1–1 : 1000 (Fig 11B) Also, pure HDL was found to be endotoxin-free as deduced from the low values in the LAL test which nearly correspond to the values of pure water
D I S C U S S I O N
The results from the biophysical measurements of the bindingof HDL to endotoxins indicate a stronginteraction, which manifests in a bindingto the lipid A backbone, in particular to the diglucosamine-phosphate region (Fig 1),
to an increase of the phase transition temperature of the acyl chains of the endotoxins and a drastic increase in acyl chain order, i.e a rigidification of the endotoxin aggregates (Fig 2) As the ms(CH2) signal results from both, the acyl chains of LPS and of the phospholipids from the HDL particles, a pure addition of the signals would lead to a curve somehow in between those of pure HDL and pure LPS, i.e
it would indicate (Fig 2) a fluidization of LPS The observation of the rigidification therefore can be assumed to
be even stronger than found in Fig 2 due to the superpo-sitions of the HDL and phospholipid signals The phase transition enthalpy of LPS with DH¼ 38 kJÆmol)1 is slightly larger compared with the data reported for phos-pholipids [10], but consideringthe difference in the number
of acyl chains (six for LPS instead of two for phospholipids),
it is strongly reduced for LPS as compared with saturated phospholipids The decrease of the phase transition
Fig 10 LPS-induced IL-6 production of human mononuclear cells by
10 ngÆmL)1LPS Re and at different [LPS]/[HDL] weight ratios was
determined in three types of examinations Preincubation of the cells
with LPS Re (30 min at 37 C) and followingaddition of HDL,
pre-incubation of the cells with HDL and subsequent addition of LPS Re,
and incubation of the cells with [LPS Re]–[HDL] complexes.
Fig 11 Endotoxin activity in the chromogenic Limulus amebocyte lysate assay at two LPS Re concentrations (100 pgÆmL)1 and
1 ngÆmL)1) and different HDL weight ratios.
Fig 9 Quotient of the fluorescence intensity at 531 nm of doubly
labeled liposomes from PtdSer vs time After incubation with LPS Re
and subsequent addition of LBP (A), after incubation with HDL and
subsequent addition of LPS Re and LBP (B), after incubation with
LPS Re and subsequent addition of HDL and LBP (C), and after
incubation with a preincubated mixture of LPS and HDL and
sub-sequent addition of LBP (D).
Trang 9enthalpy by approximately 22% at a weight ratio of [LPS]/
[HDL] 1 : 1 (Fig 3) indicates the significant influence of the
acyl chain moiety of LPS in the interaction which might be
connected with the observation that the acyl chain melting
in the presence of HDL does not take place completely This
may be taken from the wavenumber values of the LPS–
HDL sample above Tc(Fig 5) which are by 0.3 cm)1lower
than the pure LPS sample, whereas the wavenumber values
in the gel phase below Tcare more or less the same
Beside the lipid A phosphate groups as target structures
(Figs 1, 4, 5 and 8) the change of the phase transition of
dephospho LPS (Fig 4) and the remaining zeta potential
after bindingsaturation (Fig 8) give a hint that HDL
binds also to other target structures in the endotoxins, for
example to the sugar part of the endotoxins as deduced
from the band intensity decrease of the diglucosamine ring
mode (Fig 1) This interpretation is strongly supported by
the biological data: Coincubated (LPS + HDL)
com-plexes lead in both test systems to a significant decrease of
the signals (IL-6 production and LAL coagulating
acti-vity) It has been reported for synthetic endotoxins that
LAL activity is highest for preparations with a
digluco-samine backbone includingthe 4¢-phosphate (compound
504), whereas the sample without 4¢-phosphate but with
1-phosphate (compound 505) was less active by one order
of magnitude [23] Thus, the binding of HDL to LPS
must comprise at least the diglucosamine backbone
inclusive the 4¢-phosphate (see also Fig 1) which inhibits
the activity in the LAL at all concentrations (Fig 11) In
previous papers, we have reported that bindingof various
proteins (hemoglobin, lactoferrin, recombinant human
serum albumin) lead to systematic changes (increase or
decrease in dependence on the protein) in cytokine
induction, but there was no correspondingbehavior in
the LAL test [9,15,21] This can now be interpreted as
resultingfrom different target structures (epitopes) of the
proteins as found here for HDL
Concomitant with the bindingof HDL to the endotoxins,
a reorientation of the lipid A aggregate structure from
inverted cubic [21] to a multilamellar one (Fig 6), and a
slight change of the secondary structure of HDL from a
mixed a-helical/b-sheet to a predominantly a-helical
struc-ture (Fig 7) take place From this, a model of the LPS–
HDL interaction can be deduced The bindingof HDL
takes place essentially to the diglucosamine sugar backbone
and the 4¢-phosphate of lipid A The binding-places within
the HDL moiety at present cannot be given A further
possibility of LPS bindingto HDL, an incorporation of the
LPS bilayer into the HDL interior, the phospholipid bilayer
[24], does not seem to be probable as this would not explain
the data for the 1-phosphate and diglucosamine groups as
bindingsites as well as the rigidification of the acyl chains
Still unclear is the bindingstoichiometry of the LPS–
HDL system The data from the zeta potential (Fig 8)
indicate a value around 1 : 1 weight ratio At this ratio,
from an estimate of the molecular weights some hundreds
LPS molecules per HDL apolipoprotein can be calculated
This is, however, far below saturation Isothermal titration
calorimetric (ITC) experiments showed up to a weight ratio
of 1 : 6 [LPS]/[HDL] still no saturation This is also in
accordance to the biological data that a high excess weight
ratio of HDL to LPS is necessary for a saturation of the
tumor necrosis factor-a production (Fig 10)
The comparison of the results of the interaction of LPS with HDL to those published for the interaction of the former with another serum protein, albumin, indicates a completely different characteristic: Albumin (in its recom-binant form) compensates the phosphate charges to an only very low degree, the zeta potential remains lower than )40 mV, which seems to be connected with the observation that albumin does not reduce the immunostimulatory activity of LPS, rather a slight increase is observed [9] Together with the data for hemoglobin, for which also no bindingto the phosphate groups of LPS and no reduction of the immunostimulatory activity can be found [21], it may be hypothesized that a basic prerequisite for a decrease of the endotoxicity of LPS is the neutralization of the its negative charges
The bindingprocess of HDL to the endotoxins is accompanied by a dramatic decrease of the LPS immu-nostimulatory activity which is strongest when HDL is added before LPS to the cells (Fig 10) One possible explanation is the change of the aggregate structure from
a mixed unilamellar/cubic into a multilamellar one (Fig 6) In the former structures, the binding structures (epitopes) may be accessible to proteins Within the multilamellar stacks, in contrast, the epitopes of the endotoxins are more or less hidden, thus leadingto a considerable decrease of interactingmolecules such as LBP, soluble (s) or membrane-bound (m) CD14 (sCD14 and mCD14), or other receptor proteins on the cell surface [25–30] Another pathway, however, is also probable HDL by itself incorporates into phospholipid liposomes (Fig 9B,C) which is also valid for the [LPS]– [HDL] complex (Fig 9D), that means there is some similarity to the action of LBP [17] After incorporation of these molecules into target membranes, a process which is also enhanced by the action of LBP (Fig 9A), the decrease of cell activation may be understood in the light
of our conformational concept [31] Only those LPS with
a conical shape of their lipid A moiety, correspondingto
an inverted (cubic, HII) aggregate structure, represent a sufficiently high sterical stress at the site of a signaling protein such as the ion channel Maxi K [32] to induce cell signaling and, with that, cytokine induction LPS with a lipid A moiety havinga cylindrical shape are not able to induce this stress They are therefore agonistically inactive, but may block the action of active endotoxins by occupyingthe binding-sites [33] Accordingto this model and the present data, the reaggregation of the lipid A moiety due to HDL bindingfrom a cubic into a multi-lamellar structure would correspond to a change from a conical into a cylindrical molecular conformation, and would thus explain the loss of its ability to induce cytokine production
A C K N O W L E D G M E N T S
We are indebted to C Hamann, and U Diemer for performing fluorescence spectroscopic and Limulus amebocyte lysate measure-ments, respectively The expert help of B Fo¨ltingfor performingthe differential scanningcalorimetry experiments is kindly acknowledged.
We thank S.D Carroll (XOMA Corporation, Berkely, CA, USA) for the kind gift of LBP.
This work was financially supported by the Deutsche Forschungsg-emeinschaft (projects Br 1070/3–1 and SFB 367/B8).
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