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J Biol Chem, Vol. 274, Issue 42, 29624-29627, October 15, 1999
From the Polymyxin B (PMB), a cyclic cationic peptide
antibiotic, despite its severe side effects continues to occupy a
premiere position for treating endotoxicosis. Its mode of
neutralization of endotoxin has remained elusive for the last three
decades. Several synthetic peptide mimics of PMB, capable of binding
endotoxin, have been made. However, the binding ability alone appears
to be a deceptive indicator of endotoxin neutralizing activity as
molecules with similar binding propensities could either sequester or
opsonize the toxin. Hence identification of additional physical
parameters which describe adequately the outcome of PMB-endotoxin
interaction become imperative. Surface plasmon resonance (SPR) studies
reported here show that several mimics of PMB despite exhibiting
lipopolysaccharide binding affinities comparable with it but, unlike
it, do not sequester the endotoxin. These studies thus provide a
striking illustration of the difference in the behavior of PMB,
vis a vis its mimics toward the endotoxin lamellae, and
define further, in chemical terms, mechanism of the action of PMB and
allow us to posit that the design of molecules as effective antidotes
for sepsis should incorporate the ability to sequester endotoxin specifically.
Release of miniscule (nanomolar) quantities of lipopolysaccharide
(LPS),1 the major structural
component of the Gram-negative bacterial outer membrane, in systemic
bacterial infections frequently leads to a relatively common but often
fatal constellation of symptoms termed as the endotoxic shock (1, 2).
The treatment for endotoxic shock which is characterized by deranged
hemodynamics, coagulation abnormalities, and multiple system organ
failure, continues to remain nonspecific and supportive because of the absence of specific interventional strategies (3). However, the
mechanisms by which endotoxin (LPS) acts on the target cells are
increasingly being understood, which in turn has led to the development
of several experimental approaches for treating endotoxicosis. These
include sequestration of LPS by peptides or anti-LPS antibodies (4-7),
use of its antagonistic homologs that prevent its binding to the target
cells (8), or the molecules that abrogate signaling to the pathways
leading to the production of inflammatory cytokines such as tumor
necrosis factor, interleukin-1, etc. (9). Despite these many
interventional modalities and the severe side effects associated with
the use of polymyxin B (PMB), a cyclic cationic peptide antibiotic, for
the treatment of endotoxicosis, it continues to occupy the premiere
position in our armamentarium for combating endotoxicosis (3, 4). Only
recently have its mode of interaction with LPS and the structural
features involved therein been elucidated (6, 10-12). These and
earlier studies have led to the proposal that the asymmetric
distribution of the basic and nonpolar groups in polymyxin B impart to
it an amphiphilicity that is both necessary and sufficient for its LPS
neutralizing activity. That this is indeed the case was proven by a
subsequent study with a synthetic linear peptide which exhibits no
structural similarity to PMB and in which lysines and nonpolar residues
were segregated at either ends of the molecule, endowing it with
amphiphilicity sufficient for relatively strong binding to LPS (13).
Despite these seeming similarities in the mode of interaction between
PMB and other peptides with LPS, binding ability alone appears to be a
deceptive indicator of endotoxin neutralizing activity as molecules
with nearly the same binding propensities could either opsonize or sequester the toxin (14, 15). It, therefore, becomes imperative to
identify alternate or additional physical parameters of interaction which may adequately describe the outcome of the recognition of LPS on
its biologic activity, as they may aid in the design and screening of
molecules with anti-endotoxic activity. These studies are motivated by
such a consideration.
Surface Plasmon Resonance (SPR) studies reported here show that several
mimics of PMB despite exhibiting LPS binding affinities comparable with
it but, unlike it, do not sequester the endotoxin. We, therefore,
consider the removal of endotoxin as a good descriptor of the efficacy
of the anti-endotoxic activity of a given compound and suggest
inclusion of this criteria in the design and screening of
anti-septics.
Materials--
PMB sulfate, polymyxin B nonapeptide (PMBN), and
fluorescein-labeled lipopolysaccharide (Escherichia coli,
55:B5) were obtained from Sigma. Diphosphoryl lipid A was a product of
List Biologicals. Fluorescein-labeled LPS (FITC-LPS) repurified on a
Sephadex G-200 column had 8 µg of fluorescein/mg of LPS and exhibited
an emission maximum at 520 nm (excitation 495 nm). Wang resin, Opfp,
Fmoc derivatives of amino acids were obtained from Nova-Biochem. All other chemicals used were of the highest purity available.
Peptide Synthesis--
The peptides were synthesized on a
NovaSym solid-phase peptide synthesizer using standard Fmoc and Opfp
chemistry. After synthesis was completed, the terminal amino group was
deprotected from part of the resin. It was then dansylated by flowing
dansyl chloride in 10 mM triethylamine at pH 8.1 for 15 min. The peptides were cleaved from the
p-hydroxymethylphenoxy polystyrene resin using trifluoroacetic acid containing 5% water (Milli Q, Systems) and 1.5%
1,2-ethanedithiol. The sulfhydral bridge in the decapeptide was
introduced according to the method of Shih-Yi et al. (16). Peptides were purified on a reverse phase C-18 high performance liquid
chromatography column (acetonitrile:water with 0.1% TFA gradient), and
their purity was checked with a KRATOS matrix-assisted laser desorption
ionization system. The masses of the cyclic decapeptide, BPI derived
28-mer peptide, and the 23-mer peptide were 1229.9, 3407.9, and 2523.7, respectively, and their N- Quantification of Endotoxin and Peptides--
Lipid A samples
were quantified by the Limulus amoebocyte lysate assay in
pyrogen-free water, using LPS from EndosafeTM as the
standard according to the method of Yin et al. (17). Concentrations of the peptides were determined by amino acid analysis using Waters Pico-TagTM systems, whereas that of PMB and
PMBN were determined by molar absorbance and weight, respectively
(11).
Stopped-flow Spectrofluorimetry--
Fast reaction kinetic
experiments in the fluorescence mode were performed on an Applied
Photophysics SX.18MV stopped-flow apparatus equipped with ARCON 5000 RISC workstation (Leatherhead, KT227PB, UK). The dead time of the
instrument was measured to be 1.2 ms. For
N- Preparation of Liposomes, Monolayer Deposition on Alkanethiol
Chip, and Tethering of Biotinylated Dimyristoylphosphotidylethanolamine
Liposomes on Streptavidin Chip--
DMPC (1 mM) in 2:1
chloroform/methanol was dried in glass vials under nitrogen and further
dried in vacuum for 2 h. PBS, 3 ml, containing varying proportions
of lipid A (10-250 µM) was then added to each vial.
Vials were then vortexed vigorously for 10 min at 25 °C and
sonicated for 30 s in a probe sonicator. Suspension containing
liposomes were extruded about 20 times through a 50 nm polycarbonate
filters. Liposomes were separated from the unincorporated material by
passing through a 25-ml Sepharose CL-6B column. Liposomes with
biotinylated phosphatidylethanolamine and lipid A were also prepared as
above with final composition of such liposomes being 88% DMPC, 10%
lipid A, and 2% biotinylated phosphatidylethanolamine. Alkanethiol
HPATM chip was cleaned with octylglucoside, and soon
thereafter the liposomes as prepared above were passed over it at a
flow rate of 2 µl/min for 30 min and washed with PBS. Flow of
liposomes followed by PBS was repeated several times until no increase
in RUs was noted. The chip was then washed with a 5-s pulse (10 µl/min) of 20 mM NaOH to remove the multilamellar
structures to obtain stable base line. The monolayers thus obtained
were washed at a flow rate of 10 µl/min with the running buffer (PBS)
for 10 min. The surfaces are henceforth referred to as "lipid
monolayers." Lipid A (1 mg) dissolved in PBS (1 ml), heated at
60 °C for 10 min, and sonicated in a probe sonicator for 5 min was
also flown over HPA chip at 2 µl/min for 500 s and washed with
PBS for 60 s successively till a maximum of RUs was attained,
followed by washes with 20 mM NaOH till stable base line
was achieved as described above. The above surface is henceforth
referred to as "neat lipid A monolayers."
Biotinylated-phosphatidylethanolamine/DMPC/lipid A vesicles were passed
over the streptavidin sensor chip (Amersham Pharmacia Biotech) at a
flow rate of 2 µl/min for 1 h followed by a wash with PBS at a
flow rate of 5 µl/min for 20 min. These surfaces are designated as
"tethered liposomes with lipid A."
SPR Analysis--
Kinetics of the interaction of the peptides
were determined by the SPR using BiaCoreTM biosensor system
at 25 °C at a flow rate of 10 µl/min for the determination of
on-rates. Off-rates were measured in the dilution mode by flowing PBS
at 10 µl/min. The rate constants were determined by the nonlinear
least squares fitting of the primary sensogram data using the
BiaEvaluation, Version 3.0, software.
Isothermal Titration Calorimetry--
A typical titration
involved 15-20 injections at 3-min intervals, 4-µl aliquots of
peptide solution into the sample cell (volume 1.344 ml) containing
lipid A (50 µM), conducted on an OMEGA microcalorimeter of MicroCal, Inc. (11). The titration cell was stirred continuously at
400 rpm. The heat values of the dilution of the peptides in the buffer
alone were subtracted from the titration data. The resulting data were
then analyzed to determine the binding stoichiometry (n),
association constant, and the enthalpy change
( Recently we have demonstrated that the interaction of dansyl-PMB
with LPS/lipid A consists of a pair of kinetically distinguishable association and dissociation reactions (12). More specifically, the
second phase of the association reaction (k2)
was ascribed to the insertion of the hydrophobic aspects of PMB into
the nonpolar interior of the LPS lamellar phase, which as expected is
absent for PMBN which lacks the terminal
6-heptanoyl/octanoyl- Study of macromolecule-ligand interaction by SPR method depends solely
on the mass changes during the reaction (18, 19). Additionally, in SPR
the ability to form model membrane assemblies, monolayers, or bilayers
incorporating the biologic receptors which mimic the natural
environment offer additional opportunities to study, in molecular
terms, the surface-associated phenomena. A representative sensogram for
the interaction of cyclic peptide, with the monolayers of 5% lipid A
incorporated in L-
Surface Plasmon Resonance Studies Resolve the Enigmatic Endotoxin
Neutralizing Activity of Polymyxin B*
,§, and§¶
Molecular Biophysics Unit, Indian Institute
of Science, Bangalore 560 012, India and the
§ Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore 560 064, India
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-dansylated counterparts were
1258.3, 3642.8, and 2752.5, respectively. The dendrimeric peptide had a
mass of 1094.3.
-dansyl-peptide-LPS interactions, the samples were
excited at 340 nm, and emission was monitored beyond 420 nm, by using a
cutoff filter, at right angles to the excitation beam. All measurements
were made in PBS (50 mM sodium phosphate, pH 7.2, containing 150 mM NaCl) and at 20 °C (± 0.1 °C). All
traces are cumulative average of ten successive kinetic profiles.
Stopped-flow traces were analyzed for mono- and bi-exponential
reactions by curve-fitting using the Marquardt algorithm based on the
routine curves.
Hb) as described earlier.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-diaminobutyryl unit (12). Inevitably, several
peptides synthesized for countering endotoxic shock display, unlike
PMB, monophasic kinetic behavior as exemplified by the stopped-flow
kinetic analyses of the binding of the N--cyclic
decapaptide (Fig. 1, Table
I, and Scheme
1).
View larger version (22K):
[in a new window]
Fig. 1.
SPR sensograms and the stopped-flow
spectrofluorimetry of cyclic peptide-lipid A interaction.
A, monolayer of lipid A (5%) in DMPC matrix on the HPA
surface was formed as described in the text (17). B,
sensograms indicate the association and dissociation phases of the
reactions when cyclic peptide (500, 400, 300, 200, 100 nM,
top to bottom) was flown at 10 µl/min over the
monolayers containing 5% lipid A. Cyclic peptide did not bind
nonspecifically to the DMPC monolayers that lacked lipid A (data not
shown). The fits yield k1 and
k 
1 as 4.7 × 104
M1 s1 and 0.097 s1, respectively. The sensograms fitted to the mass
transport limited kinetic analysis also yielded similar results.
C, a stopped-flow trace of dansyl-cyclic peptide (10 µM) binding to lipid A (1 µM). The fit of
the data to a monoexponential reaction yields k1
of 5.1 × 104 M1
s1. Inset, the dissociation reaction
(k1 = 0.131 s1) and the plot of
kapp versus concentration of
dansyl-cyclic peptide. The Y intercept of the
kapp versus concentration yields
k1 of 0.19 s1.
Kinetic and thermodynamic parameters for the interaction of peptides
with lipid A determined by SPR, stopped-flow spectrofluorimetry, and
FITC at 20 °C
1
and 2.3 × 106 M1, respectively.
k1 and k1 of I and II and the
lipid A reported here are close to those determined by passing the LPS
over the peptides (1 and 2) immobilized covalently on CM5 chip (21).
ND, not done.
View larger version (16K):
[in a new window]
Scheme 1.
-phosphatidylcholine, dimyristoyl
(DMPC) matrix show rapid increase in RUs (Fig. 1). These changes in RUs
correspond to k1 and k1
of 4.7 × 104 M1
s1 and 0.097 s1, respectively. Residuals
confirm further the monoexponential nature of the reaction (data not
shown). The values of k1 and k1 for the dansyl-cyclic peptide-lipid A
interaction determined by the stopped-flow method and shown as a
representative example are 5.1 × 104
M1 s1 and 0.131 s1, respectively. Good agreement between the rate
constants determined by SPR with those obtained by the stopped-flow
experiments lends further confidence to our SPR data (Fig. 1 and Table
I). Unexpectedly, however, the SPR analyses of the binding of PMB with
lipid A/DMPC monolayers showed a time- and
concentration-dependent diminution in the RUs (Fig.
2, A). The
time-dependent drop in RUs indicates that PMB is able to
"take off" some mass from such monolayers. Experiments with
monolayers made with DMPC alone did not show any changes in RUs ruling
out the interaction of PMB with the phospholipid and its sequesteration
from the surface. Conversely, a time- and PMB
concentration-dependent drop in RUs from neat lipid A
monolayers was also observed (Fig. 2B). These experiments thus leave us with the inescapable conclusion that, as opposed to other
peptides examined, PMB is able to from a specific complex with
endotoxin and sequester it. Our failure to observe the initial rise for
PMB could be related to alteration of the chemical environment of the
surface which causes the signal to drop because of a conformational change in the immobilized surface. Nevertheless, appearance of FITC-LPS
in the flow-through of SPR experiments done with PMB and not in
experiments with other peptides is consistent with the removal of the
endotoxin from the immobilized surface (data not shown).
View larger version (21K):
[in a new window]
Fig. 2.
Take off of the lipid A from lipid A/DMPC
monolayers from the chip by PMB. A, PMB flown (10 µl/min) over these monolayers "lifts off" lipid A as a function
of concentration (curves 1-4 are with 2, 5, 10, and 25 mol
% of lipid A, respectively, in DMPC). PMB is also able to remove lipid
A in a concentration-dependent manner (curve 5,
PMB = 125 nm; and curve 6, PMB = 300 nm) from a
neat monolayer formed entirely of lipid A. The apparent rate constants
for the removal of lipid A from the DMPC-containing, as well as the
neat monolayers, is 0.042-0.049 s 1. B,
removal of lipid A from the tethered liposomal bilayer. The peptides
were injected over the tethered liposomes (containing 10% lipid A)
which yielded k1 and k1
values similar to the monolayer experiments. 28-residue peptide binds
to lipid A and displays typical association and dissociation reactions
(b), whereas PMB takes off the lipid A from liposomes with
similar rates as that from the monolayers of DMPC/lipid A
(c). Experiments (1-4) were conducted as a function of 25, 50, 75, and 100 nm of PMB, respectively.
We have also used liposomal preparations containing lipid A (10%)
tethered to streptavidin immobilized on the biosensor chip through
biotinylated phosphatidylethanolamine (2%) in 88% DMPC and noted
that, among the peptides studied, PMB alone manifests a drop in RUs
from such bilayer vesicles (Fig. 2A). This decrease in RUs
as a function of time reflects a specific removal of the endotoxin from
the vesicles as the liposomes without endotoxin fail to suffer any
diminution in RUs. The apparent rate constant for the removal of the
endotoxin from the neat lipid A, DMPC/lipid A monolayers and from the
tethered liposomes with lipid A are nearly the same (0.042-0.046
s1), implicating similar mechanisms in a variety of lipid
assemblies. The only difference between PMB and PMBN pertains to an
absence of the 6-heptanoyl/octanoyl diaminobutyryl group at the amino terminus of the latter. Consequently, PMBN is able to associate only at
the interfacial regions of the LPS lamellar phase while PMB, subsequent
to such an interaction, is able to penetrate the lamellar assembly
primarily through its hydrophobic region at its amino terminus (12,
20). This difference alone appears to be responsible for the poor
anti-endotoxic activity of PMBN. The cyclic (III), the dendrimeric
(IV), the 23-mer anti-endotoxic peptide (V) designed by
us,2 and the 28-mer peptide
derived from Bacterial Permeability Increasing (BPI) protein (VI) all
lack this important property and hence are unlikely to replace PMB in
clinical settings.
The mode of endotoxin-neutralizing activity of PMB is poorly understood. There are three possible ways by which it can do so: (i) by altering the organization of the endotoxin in the lamellar phase, (ii) by coating the LPS lamellar phase, and/or (iii) by solubilizing and removing it from the LPS assembly. Our SPR studies show clearly that PMB is very effective in solubilizing endotoxin from its assembly. Conversely, a number of peptides that interact with the endotoxin fail to do so.
At this stage it is instructive to consider these SPR data in conjunction with the analyses of PMB-lipid A/LPS interactions by stopped-flow spectrofluorimetry (12). As alluded to earlier, the stopped-flow experiments explicitly show that the bi-molecular association between PMB and LPS is followed by the insertion of the hydrophobic parts of the antibiotic in the apolar millieu of the endotoxin lamellar phase which, as expected, is described well by a unimolecular reaction. It, therefore, appears likely that the endotoxin "take off" in SPR experiments is synonymous mostly, if not entirely, with the unimolecular phase of the spectroscopically monitored reaction, whereas the bimolecular phase of the spectroscopically determined reaction (k1) is kinetically indistinguishable from that determined by SPR by monitoring the interaction between lipid A and the covalently immobilized PMB (21). This also appears quite likely from the fact that, under a variety conditions (such as the variation of PMB concentration), the rate of the removal of endotoxin from lipid A/DMPC and or neat lipid A monolayers or the tethered liposomal bilayers remains invariant.
In summary, these studies provide a striking illustration of the
difference in the behavior of PMB vis a vis its mimics
toward the endotoxin lamellae and define further, in chemical terms, mechanism of the action of PMB which allows us to posit that the design
of molecules as effective antidotes for sepsis should incorporate the
ability to remove endotoxin specifically.
| |
FOOTNOTES |
|---|
* This work has been sponsored by Program Support of the Department of Biotechnology, Government of India (to A. S.) under its drug and molecular design program. This work was carried out in the BiaCore facility funded by the Department of Biotechnology, Government of India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 91-80-309-2714; E-mail: surolia@mbu.iisc.ernet.in.
2 C. J. Thomas and A. Surolia, unpublished work.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
LPS, lipopolysaccharide;
PMB, polymyxin B;
SPR, surface plasmon resonance;
PMBN, polymyxin B nonapeptide;
FITC, fluorescein isothiocyanate;
Fmoc, N-(9-fluorenyl) methoxycarbonyl;
PBS, phosphate-buffered saline;
DMPC, L-
-dimyristoylphosphatidylcholine;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
RU, response unit(s).
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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C. J. Thomas, N. Surolia, and A. Surolia Kinetic and Thermodynamic Analysis of the Interactions of 23-Residue Peptides with Endotoxin J. Biol. Chem., September 14, 2001; 276(38): 35701 - 35706. [Abstract] [Full Text] [PDF]
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