(Received for publication, March 8, 1995; and in revised form, May 4, 1995)
From the
Endotoxic shock follows a cascade of events initiated by release
of lipopolysaccharide during infection with Gram-negative organisms.
Two overlapping 15-mer peptides were identified, corresponding to
residues 91-108 of human lipopolysaccharide binding protein that
specifically bound the lipid A moiety of lipopolysaccharide with high
affinity. The peptides inhibited binding of lipopolysaccharide to
lipopolysaccharide binding protein, inhibited the chromogenic Limulus
amebocyte lysate reaction, and blocked release of tumor necrosis factor
Infection with Gram-negative organisms results in the specific
activation of mononuclear phagocytes by the bacterial cell wall
constituent lipopolysaccharide (LPS) LPS stimulation of
mononuclear cells is driven by the cellular LPS receptor CD14 (3, 4) and is potentiated as much as 1000-fold by
prior complexing of LPS with the serum protein LBP(5) . Because
serum LPS concentrations found in the clinical setting are very
low(3) , it appears that LPS toxicity largely depends on the
presence of LBP, a hypothesis that has been borne out in in vivo models of LPS toxicity(6) . Thus, we were interested in
better understanding the different structure-function relationships
underlying the formation of specificity of LBP for LPS. Here, we report
the identification of an LPS binding site of LBP, determined by
scanning the entire primary structure of LBP for the presence of small
LPS binding domains. Only one domain, corresponding to
LBP-(91-108), was found to bind LPS tightly. This region most
likely comprises at least part of the LPS binding site of LBP.
Preparative scale peptide syntheses
were performed essentially as previously described (7) on
4-methyl-benzhydrylamine resins with an ABI 431A synthesizer using
version 1.12 of the standard Boc software. Following HPLC purification,
all peptides had the correct amino acid analysis and molecular ion
(fast atom bombardment mass spectroscopy or plasma desorption mass
spectroscopy) and were greater than 95% pure as determined by two
different HPLC gradients. Peptide sequences are given in the one
letter code, with the following modifications: Ac- denotes N-acetylated derivatives, -NH
LBP was released from the LBP-Ig by limited proteolysis
with papain, and the immunoglobulin constant regions were removed by
passage over a protein A affinity column. The released LBP (LBP60k)
appeared as a 60-kDa band that was indistinguishable from native LBP by
SDS-polyacrylamide gel electrophoresis. This material was subsequently
biotinylated using D-biotin-N-hydroxysuccinimide
ester following the manufacturer's instructions (Boehringer
Mannheim).
An ELISA-based competitive inhibition assay was used to
detect LPS binding peptides by means of their ability to inhibit
binding of LPS-horseradish peroxidase to LBP-Ig. Since LBP binds
different forms of LPS via the common lipid A moiety N,O-acylated We synthesized a panel of 146 overlapping 15-mer
peptides corresponding to the entire LBP primary structure (10) and screened the crude peptides for LPS blocking activity
in the competitive inhibition assay. Interestingly, only two of the
nested peptides, 31 (WKVRKSFFKLQGSFD-NH
Figure 1:
Inhibition of LPS-horseradish
peroxidase binding to Fc-captured LBP. A, inhibition of
LPS-horseradish peroxidase binding to Fc-captured LBP by LBP-related
nested peptides. Each data point denotes the OD found in the
competitive ELISA for the indicated peptide; all peptides tested at 150
µM final concentration as described under ``Materials
and Methods.'' Nested peptide number denotes the 15-mer peptide
starting with the mature LBP residue position equal to (3n)
- 2, where n = the nested peptide number. Thus,
peptide number 31 = LBP residues 91-105. Data represent
two separate assays. Background signal not subtracted. B,
competitive inhibition of LPS-horseradish peroxidase binding to
Fc-captured LBP-Ig by LBP-(91-108)-related peptides. Indicated
concentrations of LBP-related peptides coincubated with LPS-horseradish
peroxidase in the presence of Fc-captured LBP-Ig. Closedsquares correspond to LBP-(91-105)-NH
Three peptides from
the LBP-(91-108) region were synthesized and purified to
homogeneity: LBP-(91-105)-NH To define further the
specificity of the LBP-(91-108)-related peptides for LPS, two
types of preincubation experiments were carried out. First, as
expected, preincubation of unlabeled LPS with Fc-captured LBP-Ig,
followed by washing, inhibited subsequent binding of LPS-horseradish
peroxidase (Fig. 2A). However, preincubation of the
peptides with Fc-captured LBP-Ig, followed by washing, had no effect on
subsequent binding of LPS-horseradish peroxidase; this indicated that
the peptides do not interact with LBP-Ig to inhibit LPS binding.
Second, binding of biotinylated LBP (LBP-B) to LPS-coated plates could
be inhibited in a dose-dependent manner by preincubation of the plates
with various analogs of LBP-(91-108) (Fig. 2B) or
by polymyxin B. As before, LBP Ac-(91-105)-NH
Figure 2:
Inhibition of LBP binding of LPS by
preincubation of LBP-related peptides. A, indicated
concentrations of inhibitors (opencircles correspond
to LBP-(91-108)-NH
We calculated the LPS binding
affinity of the LBP-(91-108) peptides after determining the
relative ability of the LBP peptides and polymyxin B to compete with
LBP-B for binding to solid phase LPS. The signal obtained from binding
8 nM LBP-B to solid phase LPS was inhibited, by 50%, by
simultaneous addition of 1 µM polymyxin B, indicating a
125-fold difference in relative affinity for LPS. Since polymyxin B has
a K
Two models were utilized to determine if the
LBP-(91-108) peptides could also block complex biological
responses to LPS challenges in vitro. The chromogenic limulus
amebocyte lysate assay (LAL) is sensitive to minute amounts of LPS (as
little as 1 pg/ml) and is specific for smooth and rough LPS forms, as
well as for lipid A. It can be seen (Table 2) that the same
LBP-(91-108) peptides that were found to block binding of LPS by
LBP also can neutralize the LAL reaction to lipid A;
LBP-(91-105)-NH
A large degree of LPS toxicity is a
consequence of TNF
Figure 3:
LBP-(94-108)-NH
The LBP-(91-108) peptides were also
tested for the ability to block TNF
Figure 4:
Ability of LBP-related peptides to block D(+)-galactosamine-potentiated TNF
Few examples exist in the literature
that describe potent blocking peptides with K The
results suggest that in the intact protein, LBP residues 91-108
play a critical role in the formation of specificity to LPS, forming at
least part of the LPS binding site. This proposal is supported by
several previously reported observations. (i) Recent modeling
experiments have attempted to predict the LPS binding site of human LBP
based on the three-dimensional crystal structure of the
Limulus-anti-LPS factor and certain sequence similarities between
Limulus-anti-LPS factor and LBP(14) . Interestingly,
LBP-(91-108) comprises the loop and the second
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding
protein residues 91-108 form at least part of the
lipopolysaccharide binding site. Moreover, derivatives of
lipopolysaccharide binding protein residues 91-108 might modulate
lipopolysaccharide toxicity in the clinical setting.
(
)(reviewed
in (1) ). Overstimulation with LPS, a frequent consequence of
Gram-negative sepsis, can result in systemic flooding with potent
proinflammatory cytokines that include interleukin-1, interleukin-6,
and tumor necrosis factor
(TNF
). This contributes directly
to the development of endotoxic shock, multiple organ failure,
disseminated intravascular coagulopathy, and acute respiratory distress
syndrome; these are collectively referred to as Gram-negative sepsis
syndrome. More than 120,000 cases of Gram-negative sepsis syndrome
occur in the United States annually(2) .
Peptide Synthesis
An Advanced ChemTech
model 350 peptide synthesizer was used for the simultaneous synthesis
of up to 96 peptides. The double coupling FMOC (N-(9-fluorenyl)methoxycarbonyl) protocol was followed, and
synthesis products were worked up according to the manufacturer's
specification. Crude nested peptides were screened for LPS blocking
activity as described below. denotes
COOH-terminal amidated derivatives, and d- denotes D-isomer substitution of the amino-terminal residue only.
Recombinant LBP
The LBP-immunoglobulin
fusion protein was comprised of human LBP coding sequences fused to
human IgG1 CH1 sequences and secreted from SP2/O murine myeloma cells
in association with a human Ck domain from which the Vk region had been
deleted. The LBP-Ig was purified to homogeneity by means of protein A
chromatography. Acid (0.1 M citrate, pH 3.5)-eluted material
was immediately neutralized with a molar excess of pH 7.8 Tris buffer
and dialyzed extensively against PBS. Only material with low endotoxin
content (<10 enzyme units/ml), as measured by the chromogenic
Limulus amebocyte lysate assay (Bio Whittaker), was used in subsequent
studies.LPS-Horseradish Peroxidase
Conjugate
LPS-horseradish peroxidase conjugate was obtained
from Alercheck, Inc. (Portland, ME). According to the manufacturer, it
was prepared from Escherichia coli J5 LPS (rough LPS) by
direct coupling of labeling grade horseradish peroxidase (Boehringer
Mannheim) to periodate-activated LPS. The horseradish peroxidase:LPS
coupling ratio was 2.5:1.Competitive Inhibition of LPS Binding to
LBP
LBP-Ig was Fc-captured by coating ELISA plates with 10
µg/ml goat anti-human Fc antibodies (Jackson ImmunoResearch) in
bicarbonate buffer. After washing, purified LBP-Ig was added to the
wells at 1 µg/ml in PBS containing 1% nonfat dry milk (PBSM), and
allowed to incubate at room temperature for 30 min. Following washing,
individual competitors and an equal volume of 250 µg/ml
LPS-horseradish peroxidase were added to the ELISA wells and allowed to
incubate at room temperature for 1 h. The plates were washed with PBS,
and the chromogenic substrate O-phenylenediamine
dihydrochloride was added and processed as described by the
manufacturer (Sigma). In experiments using the preincubation format,
the inhibitors are added alone to the assay wells and allowed to
incubate prior to washing and addition of LPS-horseradish peroxidase.Competitive Inhibition of LBP Binding to
LPS
For ELISA assays measuring the binding of biotinylated
LBP to solid phase LPS, ELISA plates were coated with 50 µg/ml E. coli 0111:B4 LPS in bicarbonate buffer essentially as
described(8) . After washing with PBSM, different inhibitor
dilutions (in PBSM) were incubated alone (preincubation experiments) or
with an equal volume of 1 µg/ml (in PBSM) biotinylated LBP
(competitive inhibition) and were incubated for 1 h at room
temperature. For the preincubation assays, the plates were further
washed, and 0.5 µg/ml (8 nM in PBSM) biotinylated LBP
(LBP-B) was incubated and washed as before. Bound LBP was detected in
both formats with 1/1000 Z-avidin-horseradish peroxidase conjugate
(Zymed) and processed as described by the manufacturer.LPS Stimulation of PBMC
Human PBMC were
purified from normal donor blood by differential centrifugation using
mono-poly resolving medium (Flow-ICN) and following the instructions of
the manufacturer. In a 200-µl final volume of Iscove's
serum-free medium (JRH Biosciences), 5 10
PBMC were
combined with different concentrations of E. coli 0111:B4 LPS
and inhibitor peptides. LBP potentiated cultures were supplemented with
200 ng/ml (3 nM) purified human LBP (LBP60k, described above).
Cultures were incubated for 3 h in a 37 °C humidified CO
incubator; supernatants were collected and assayed for human
TNF
content by means of the previously described WEHI
bioassay(9) . Purified human recombinant TNF
(Genzyme) was
used as standard.
1,6-D-glucosamine
disaccharide 1,4`-bisphosphate(8) , this competitive inhibition
ELISA could potentially identify lipid A-specific peptides. Indeed, we
found that LBP-Ig can specifically bind LPS-horseradish peroxidase in a
way that is inhibitable by different LPS- and LBP-specific blocking
molecules:
(
)(i) smooth (E. coli 0111:B4) and rough (E. coli J5) unlabeled LPS and lipid A (Salmonellaminnesota R595) could competitively
inhibit binding of LPS-horseradish peroxidase to the Fc-captured
LBP-Ig, each reaching 50% inhibition in the 10 µM range;
(ii) polymyxin B (Roerig), a potent lipid A binder, also could inhibit
the LPS-horseradish peroxidase with an IC
of 0.05
µM; (iii) 8 of 26 different anti-human LBP monoclonal
antibodies were found to inhibit the binding of LPS-horseradish
peroxidase to LBP-Ig; (iv) a control LBP-Ig fusion protein in which the
COOH-terminal-half of the LBP domain had been deleted displayed no LPS
binding activity.
), corresponding to
LBP residues 91-105, and 32 (RKSFFKLQGSFDVSV-NH
),
corresponding to 94-108, could completely inhibit binding of
LPS-horseradish peroxidase by LBP-Ig (Fig. 1A). The two
active peptides were selected for further study.
; opencircles correspond to
LBP-(93-108)-NH
; opensquares correspond to LBP-(91-108)-NH
; closedcircle corresponds to
LBP-(Ac-91-105)-NH
. Background signal was not
subtracted.
(WKVRKSFFKLQGSFD-NH
), LBP-(94-108)-NH
(RKSFFKLQGSFDVSV-NH
), and LBP-(91-108)-NH
(WKVRKSFFKLQGSFDVSV-NH
). All three peptides were
freely soluble in distilled water. The peptides were assessed for the
ability to block binding of LPS-horseradish peroxidase to Fc-captured
LBP-Ig as before and were found to exhibit similar IC
values, each in the 10-20 µM range (Fig. 1B). A fourth analog was synthesized,
LBPAc-(91-105)-NH
(Ac-WKVRKSFFKLQGSFD-NH
); interestingly, it was found
to be totally inactive, at least within the concentration range tested.
This relatively inactive analog was used as a negative control in
several of the experiments described below.
did not
block binding of LBP to LPS. These results show that the
LBP-(91-108) peptides specifically inhibit the LPS-LBP
interaction by binding to LPS.
; opensquares correspond to LBP-(91-105)-NH
; closedsquares correspond to LBP-(94-108)-NH
; opentriangles correspond to S. minnesota R595 lipid A, M
1700; and closedtriangles correspond to E. coli 0111:B4 LPS, 25,000 average molecular weight) preincubated with
immobilized LBP-Ig. Following washing, LPS-horseradish peroxidase was
added and ELISA was processed as described under ``Materials and
Methods.'' B, indicated concentrations of inhibitors (opensquares, LBP-(91-105)-NH
; closedsquares, LBP-(94-108)-NH
; closedtriangles, polymyxin B; opentriangles, LBP-(Ac-91-105)-NH
; and opencircles, LBP-(91-108)-NH
)
preincubated with E. coli 0111:B4 LPS-coated ELISA plates.
Following washing, LBP-B was incubated and then washed and probed with
horseradish peroxidase-conjugated streptavidin. Background signal was
not subtracted.
= 8.6
10
M, the relative affinity of LBP-B for LPS = (8.6
10
)/(125) = 6.9
10
M, in good agreement with a previous
report(8) . Values for the peptides were calculated using the
same method and ranged between 0.1 and 0.04 of the affinity held by
polymyxin B (Table 1). Interestingly, the affinities of the LBP
peptides were found to be improved by as much as 12-fold by replacing
the amino-terminal amino acid residues with D-isomers (Table 1). Thus, the LPS binding affinities of the LBP peptides
approach that of polymyxin B, a potent but highly toxic
inhibitor(11) .
and LBP-(91-108)-NH
displayed similar lipid A neutralizing potencies as found for
polymyxin B (Table 2). These results show that the LBP peptides
can completely block the exquisitely sensitive LAL reaction to lipid A
and accordingly indicate that the LBP-(91-108)-related peptides
are specific for lipid A.
released by CD14
monocyte/macrophage lineage cells following exposure to
LPS
LBP complexes(12, 13) . Accordingly, we
wished to determine if the LBP peptides could block the LBP-dependent
LPS response of human mononuclear cells in vitro. Fig. 3shows the LBP-dependent TNF
response of purified
human PBMC to different doses of LPS and the blocking ability of a
representative LBP peptide. In the absence of human serum, PBMC are
completely unresponsive to 1 ng/ml LPS. As previously
shown(5, 10) , potentiation with added purified LBP
restored the ability of the PBMC to release robust amounts of TNF
in response to 1 ng/ml LPS. Addition of only 3 µM
LBP-(94-108)-NH
inhibited this LBP-dependent TNF
response to 1 ng/ml LPS by 50%, and 30 µM LBP-(94-108)-NH
completely abolished the
TNF
response. In fact, 30 µM
LBP-(94-108)-NH
could substantially inhibit the PBMC
response to 10 ng/ml LPS, even though much of this response appears to
be LBP independent. In separate experiments, similar results were also
obtained using a serum-stable analog of LBP-(94-108)-NH
(viz. LBP-(d-94-108)-NH
) when
tested with 1% normal human serum-potentiated PBMC response to 1 ng/ml
LPS.
The results show that 1-10 µM LBP-(94-108) peptides can block both purified LBP- and
normal human serum-potentiated LPS responses to physiologically
relevant doses of LPS.
inhibition of LBP-dependent PBMC TNF
response to smooth LPS.
Indicated concentrations of E. coli 0111:B4 LPS incubated with
PBMC are as follows: opensquares, under serum-free
conditions; opencircles, potentiated with 0.2
µg/ml purified human LBP60k; closedcircles,
LBP-potentiated and in presence of 0.3 µM LBP-(94-108)-NH
; closedtriangles, LBP-potentiated and in presence of 3.0
µM LBP-(94-108)-NH
; solidsquares, LBP-potentiated and in presence of 30 µM LBP-(94-108)-NH
. TNF
concentration
determined as described (9) following incubation for 3 h at 37
°C. Assay shown is representative of three separate
experiments.
response to LPS challenge in vivo. When mice were coinjected with 1 ng of
LPS/galactosamine and individual LBP-(91-108) peptides, each of
the LBP-(91-108) peptides could significantly inhibit the
TNF
response (Fig. 4, A-E). All of the
peptides could inhibit by at least 50%; two of the peptides,
LBP-(d-94-108)-NH
and
LBP-(d-91-108)-NH
, could completely inhibit
the TNF
response when administered at about 10 and 40
µg/mouse, respectively. Fig. 4, panelF,
shows the relative LPS neutralization potencies of the peptides. A
500-fold range of potencies was evident, with 0.1 µg/mouse of the
most potent peptide blocking by 50% the TNF
response to 1 ng of
LPS. Because the maximum serum LPS levels generally found among septic
patients do not exceed 0.1 ng/ml, the results augur well for the
ability of these peptides to inhibit LPS toxicity in vivo (studies in progress).
response to
smooth LPS challenges in vivo. In a 200-µl final volume of
PBS, indicated amounts of individual peptides were combined with 1 ng
of E. coli 0111:B4 LPS and 15 mg D(+)-galactosamine prior to intravenous injection into
6-10-week-old female CD-1 mice (Charles River). 1 h later, mice
(3/group) were sacrificed, and collected serum was assayed for TNF
content by means of the WEHI bioassay as previously
described(9) .
in the submicromolar range. This is true even when detailed
knowledge of the three-dimensional structure of the receptor and its
ligand are available. Therefore, it is remarkable that two overlapping
15-mer LPS binding peptides were identified that can completely block
the high affinity interaction between LPS and LBP. Our success with
this strategy may reflect essential differences in how protein
receptors recognize small ligands (such as the
1,6-D-glucosamine disaccharide 1,4`-bisphosphate that
most likely represents the moiety of lipid A recognized by LBP), as
opposed to the large discontinuous interactive surfaces characteristic
of protein-protein interactions. Accordingly, it is possible that the
approach described here might be of general use when the interactive
portion of the ligand is predicted to be relatively small.
-strand of
the amphipathic loop structure predicted to be the LPS binding site in
these modeling experiments. (ii) Rabbit LBP, when cleaved at a unique
plasmin hypersensitivity site between residues 99 and 100, loses all
LPS binding activity(1) . (iii) The amino-terminal fragment of
LBP (residues 1-197) retains the LPS binding activity of the
native molecule(15) . (iv) A 15-mer peptide derived from
bactericidal permeability increasing protein (BPI) has been described
that neutralizes LPS in the LAL reaction and is
bactericidal(16) , two properties of native
BPI(17, 18) . LBP and BPI display considerable
sequence identity (10) and share the ability to bind LPS with
high affinity. After sequence alignment (which does not require the
introduction of gaps through residues 122)(10) , the
bactericidal/LPS neutralizing BPI-related peptide (BPI 85-99)
shares partial identity with the active LBP oligomers described here
(LBP 91-108). However, unlike BPI(85-99), preliminary
experiments indicate that the LBP-(91-108)-related peptides do
not inhibit growth of Gram-negative organisms.
The results
described here suggest high resolution site-directed mutagenesis
experiments that, together with monoclonal antibody epitope mapping
experiments and extensive analog peptide structure-function analysis,
will lead to a more detailed understanding of the LBP-LPS interaction
(studies in progress).
, tumor necrosis factor
; PBMC, peripheral blood mononuclear cells; BPI, bactericidal
permeability increasing protein; HPLC, high pressure liquid
chromatography; PBS, phosphate-buffered saline.
We thank B. Scallon for kindly providing the LBP-Ig
and purified recombinant LBP used in these studies and Marian
Kruszynski, Robert Weber, and Margret Falcone for synthesis,
purification, and physical characterization of the LBP-related
peptides.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.