From the
Endotoxin (lipopolysaccharide; LPS) activates a wide variety of
host defense mechanisms. In mammals LPS binding protein (LBP) and CD14
interact with LPS to mediate cellular activation. Using sucrose density
gradients and a fluorescent endotoxin derivative we have investigated
the mechanism of LPS binding to LBP and the soluble form of CD14
(sCD14). LPS binds to LBP to form two types of complex; at low ratios
of LPS to LBP complexes with one molecule of LBP and 1-2
molecules of LPS predominate, while at high ratios of LPS to LBP a
large aggregate of LBP and LPS predominates. Complexes of LPS with
sCD14 do not form large aggregates, consisting of only 1-2 LPS
bound to a single sCD14 even at high multiples of LPS to sCD14. LBP
catalyzes LPS binding to sCD14. Catalysis by LBP apparently occurs
because LBP provides a pathway for LPS to bind to sCD14 which avoids
the necessity for LPS monomers in aqueous solution. The dissociation
constants for LPS
The exposure of organisms as diverse as horseshoe crabs and
mammals to endotoxin (lipopolysaccharide, LPS)
In this report we describe
the use of fluorescein-derivatized LPS (FITC-LPS) to permit continuous
monitoring of the formation of LPS
Salmonella minnesota Re595 LPS was isolated and
fluoresceinated with fluorescein isothiocyanate (Molecular Probes,
Eugene, OR) by published methods
(8, 9) . FITC-LPS from
several commercial sources was not useful for these experiments. The
LPS concentration of the derivative was determined by assay of the
2-keto-3-deoxyoctulosonic acid and measurement of the fluorescein
content by optical density at 493 nm as described
(9) . The
products used here had substitution ratios of 20-30 mol %
fluorescein. Based on the structure of S. minnesota Re595 LPS,
its molecular weight was taken to be 2300
(10) . While the
derivatization procedure used to make FITC-LPS may alter its physical
properties from the unsubstituted molecule, the FITC-LPS and the
[
Sucrose density gradients were
centrifuged for 1 h 20 min at 4 °C in a pre-chilled TV-865 rotor
(DuPont, Burbank, CA) at 55,000 rpm. The gradients were 4 ml of
5-20% sucrose in 0.05 M phosphate, 0.14 M NaCl,
2 mM EDTA, pH 7.4, layered onto 0.2 ml of 40% sucrose. The
samples were incubated at 37 °C for 15 min and then chilled to 4
°C before 0.2 ml was layered on top of the gradient; aliquots of
the samples were removed before centrifugation to determine total
radioactive material for calculation of recovery. Typically, recovery
of radioactive material from the gradients was 65-95%.
Fluorescence was monitored using an SLM 8000 fluorimeter (SLM
Instruments, Urbana, IL) with excitation at 490 nm and emission at 520
nm. In plain glass cuvettes (7 mm
On-line formulae not verified for accuracy where F is the observed fluorescence, L and P are the concentrations of FITC-LPS and protein, respectively, SQRT
is the square root operator, and
The
K
As one would expect, unfluoresceinated Re595 LPS
competes with FITC-LPS for binding to LBP (data not shown).
Furthermore, monoclonal antibody 2B5, which binds LBP and blocks LPS
binding to recombinant mCD14 expressing Chinese hamster ovary cells
(16) also blocks FITC-LPS binding to LBP (data not shown).
The kinetics of formation of FITC
Heretofore it has not been possible to monitor the
interaction of LPS with any LPS binding protein on a continuous,
quantitative basis to explore the mechanism, kinetics, and equilibria
of the reaction. While several strategems have been employed to
determine binding affinities for LPS with several binding proteins
(17, 18, 19, 20) , the kinetics of the
binding reactions have not been measurable. Through the use of
FITC-LPS, many, although not all, of the difficulties are removed. If,
as we hypothesize, the increase in fluorescence derives from relief of
fluorescein self-quenching in an FITC-LPS aggregate, then the method
will not be useful for types of FITC-LPS which do not aggregate.
However, these may be relatively few given the strongly amphipathic
nature of the lipid A moiety of LPS. Additionally, not all types of
LPS, and not all LPS partial structures, have readily derivatizable
groups in appropriate parts of the molecule. Indeed, we were unable to
observe any fluorescence change when the FITC derivative of
Escherichia coli 0111:B4 LPS bound to LBP (data not shown).
Wistrom-Aurell et al.
We conclude that LBP catalyzes the reaction of
LPS with sCD14 for the following reasons. If LBP were a stoichiometric
partner in the final complexes formed, this should be visible in the
sucrose density gradients of Fig. 2, but it is not. LBP should be
labeled by [
Two possible reaction
pathways which are potentially consistent with the kinetic data for the
reaction of aggregated FITC-LPS (FITC-LPS
Fig. 5
, b and c, suggests that as the concentration of either
protein changes, the overall reaction undergoes a change in
rate-limiting step. This is typically observed when one step of a
multistep reaction is subject to catalysis
(22) . For example,
given the foregoing discussion, in Fig. 5 c we propose
that the rate-limiting step is ``5'' at low [sCD14]
and ``1'' at high [sCD14]. Under this
interpretation, the value observed for k
Biologically, these results have several
implications. As noted previously, recent work has suggested a
catalytic role for LBP in assisting the presentation of LPS to sCD14
and that catalytic role is clearly supported by these results, as is
the conclusion that sCD14 only binds a small number of LPS molecules
(7) . While one might wish to extrapolate and suggest that LBP
also catalyzes the binding of LPS to mCD14
(7) , i.e. CD14 on monocyte or neutrophil membranes, recent results from this
laboratory show that LBP remains associated with mCD14 bearing cells
when the cells are incubated with LPS
It is also known that the in vitro activation of
peripheral blood monocytes is some 1000-fold more sensitive to LPS than
is the activation of endothelial cells and it has been proposed that
the LPS-sCD14 pathway for activation of LPS-sCD14-sensitive cells
exposed to blood is probably secondary to their activation by LPS
elicited monocyte derived cytokines
(25) . This hypothesis is
supported by the measurements of K
The fact that FITC-LPS is actually
transferred to sCD14 in our experiments despite the fact that sCD14
binds FITC-LPS some 10-fold less tightly than LBP is a result of the
fact that sCD14 was present at much higher concentration than LBP.
However, in serum (or plasma), LBP and sCD14 are normally present at
roughly equal concentrations. One would thus predict that in serum, LPS
would predominantly associate with LBP. During an acute-phase response,
LBP concentrations are considerably elevated, more so than sCD14. Thus
after an acute-phase response, the balance of LPS bound to LBP and
sCD14 would shift further to LBP, further diminishing the ability of
LPS to stimulate cells via the LPS-sCD14 pathway. Thus although an
important role for LBP catalysis of LPS binding to sCD14 is readily
demonstrable in vitro, the role of this reaction in vivo remains to be defined. Perhaps it will be found at extravascular
sites of sCD14 synthesis where the ratio of sCD14:LBP could be quite
different from plasma
(26) .
This is
publication 9103-IMM from the Scripps Research Institute.
LBP and LPS
sCD14 complexes were determined
to be 3.5
10
and 29
10
M, respectively. These numbers suggest that when LBP and
sCD14 are present at roughly equal concentrations as they are in normal
human plasma and compete for limited LPS, the LPS will predominantly
associate with LBP.
(
)
results in the activation of a wide variety of host defense
mechanisms. Recent studies of mammalian cells have described a unified
pathway for the LPS-dependent activation of many cell types including
monocytes, macrophages, neutrophils, endothelial cells, smooth muscle
cells, and some epithelial cell lines. The common elements of the
pathway include LPS binding protein (LBP), a 60-kDa plasma
glycoprotein, and CD14, a 55-kDa glycoprotein. On monocytes,
macrophages, and neutrophils, CD14 is present as a
glycerophosphoinositol tailed membrane (mCD14) component
(1) .
In the presence of LBP, LPS binds to the mCD14 and initiates cellular
activation
(2, 3) . However, other LPS-responsive cells
such as endothelial cells, smooth muscle cells, and some epithelial
cell lines do not express mCD14. Instead, they have a receptor for
complexes of LPS with the soluble form of CD14 (sCD14), which
circulates in plasma without a glycerophosphoinositol tail
(4, 5, 6) . Here again, LBP facilitates the
formation of LPS
sCD14 complexes.
LBP and LPS
sCD14
complexes. Heretofore it has not been possible to observe the formation
of LPS
LBP and LPS
sCD14 complexes on a real time basis with
a method that also permits the quantitative determination of kinetic
and equilibrium parameters for formation of these complexes. We also
describe our characterization of LPS
LBP and LPS
sCD14
complexes using sucrose density gradients. Others have used
nondenaturing polyacrylamide gel electrophoresis to characterize LPS
protein complexes
(7) , but we found sucrose density gradients
more useful since they permit more analysis of the separated
components. Taken together, we conclude from these results that LBP
catalyses the formation of LPS
sCD14 complexes and suggest a
mechanistic pathway by which this occurs.
I]ASD-LPS retain their biological activity
(data not shown). Rabbit LBP was isolated from acute-phase rabbit serum
(11) . Human recombinant soluble sCD14 was isolated by
immunoaffinity chromatography (Immunopure Protein G IgG Orientation
Kit, Pierce, Rockford, IL) using monoclonal antibody 63D3 (American
Type Culture Collection, Rockville, MD) from the supernatants of
Chinese hamster ovary cells transfected, essentially as described in
Ref. 12, with the cDNA for human CD14. To prepare
S-labeled sCD14 and
S-labeled sLBP CD14 or
LBP, expressing Chinese hamster ovary cells were metabolically labeled
with [
S]Met (DuPont NEN, Boston, MA). The
labeled proteins were prepared from 24-h culture supernatants by
immunopurification using mAb 63D3 for sCD14 and mAb 18G4 for LBP. The
mAb 18G4 as well as mAb 2B5, with specificity for human LBP, were
kindly provided by D. Leturcq and A. Moriarty (R. W. Johnson
Pharmaceutical Research Institute, La Jolla, CA).
[
I]ASD-LPS was prepared from S. minnesota Re595 LPS as described
(11) .
H-Labeled LPS and
non-radioactive LPS from Salmonella typhimurium PR122(Rc) was
obtained either from R. Munford
(13) or commercially (List
Biologicals, Campbell, CA).
45 mm, Sienco, Morrison, CO)
S-labeled LBP was observed to disappear from solution at a
significant rate, presumably by adsorption to the glass (data not
shown). In order to minimize binding of LBP and FITC-LPS to the
cuvettes, they were filled with 10 mg/ml bovine serum albumin (Sigma)
in water for 1 h after which they were extensively rinsed with water
and stored moist at 4 °C. As indicated by the slow declines of
fluorescence seen at long times in some figures, this procedure is not
totally successful. Reactions were conducted in 50 mM
phosphate, 100 mM NaCl, 2 mM EDTA at pH 7.4, except
for the experiments reported in Fig. 3 b which were
conducted in 10 mM phosphate, 2 mM EDTA, at pH 7.4
with KCl as required. Apparent pseudo-first order rate constants and
dissociation constants were obtained from the fluorescence data using
the curve fitting procedures of SigmaPlot (Jandel Scientific, San
Rafael, CA). When loss of fluorescence due to adsorption to the cuvette
was significant (see above), it was included as a time linear component
of the overall fluorescent change. In some instances the amount of
FITC-LPS approached 10% of the binding protein. Therefore, in all
cases, K
values were obtained by fitting
the data to a ``quadratic'' hyperbola rather than a
``rectangular'' hyperbola as defined by Equation 1,
F and K, the
overall fluorescence change and the dissociation constant respectively,
are the fitted parameters.
Figure 3:
a,
fluorescence intensity versus time of FITC-LPS (4.2
10
M) added at time A mixed with
LBP (4.2
10
M) added at time
B. Inset, the data from the larger figure and their
fit to a pseudo-first order reaction with k
= 4.72
10
s
.
b, the influence of salt concentration on the apparent first
order rate constant for FITC-LPS-LBP formation. c, the net
change in fluorescence upon mixing LBP with FITC-LPS (4.4
10
M). The solid line is fit to a
quadratic hyperbola with K apparent of 1.0
10
M (see ``Experimental
Procedures'' for details).
The photoaffinity probe
[I] ASD-LPS (8
10
M) was incubated with several mixtures of sCD14 and LBP
(see legend to Fig. 6) at 37 °C for 15 min before chilling to
4 °C for photolysis as described previously
(11) . The
proteins were then separated by SDS-polyacrylamide gel electrophoresis
using a 10% acrylamide gel and detected by autoradiography after
Coomassie staining to detect protein molecular weight markers (Sigma).
Figure 6:
SDS-polyacrylamide gel electrophoresis gel
analysis of [I]ASD-LPS labeled mixtures of
sCD14 with LBP at 0.3
10
M using 8
10
M [
I]ASD-LPS. The autoradiograph of the gel
is shown together with the mobilities of purified LBP, sCD14, and the
molecular weight markers bovine serum albumin ( 67) and
ovalbumin ( 43).
Sucrose Density Gradient Analysis of LPS
When LBP
Complexes
H-labeled LPS or
S-labeled LBP are separately subjected to sucrose density
gradient sedimentation, their mobilities are quite different as shown
in Fig. 1, panels a and b, respectively. The
concentrations of
H-labeled LPS and
S-labeled
LBP used, as well as the mole ratios of the incubation mixtures and the
resulting sucrose density gradient peaks, are given in . As
indicated by the mobilities of albumin and ovalbumin (data not shown),
S-labeled LBP sediments with the mobility expected for a
60-kDa protein. However,
H-labeled LPS has a mobility
considerably higher than would be warranted by its molecular weight of
4000
(13) , indicating a high degree of aggregation. When
H-labeled LPS and
S-labeled LBP are mixed and
incubated for 15 min at 37 °C before sedimentation, the mobility of
the
H-labeled LPS shifts to the mobility of LBP while the
mobility of the
S-labeled LBP does not increase
(Fig. 1, panel c). This could be the result either of
LPS binding to LBP or simply disaggregation of the
H-labeled LPS. Since [
I]ASD-LPS and
LBP can be demonstrated to form a complex at lower concentrations than
used here (see Fig. 6), we conclude that a complex of
H-labeled LPS and
S-labeled LBP has formed and
that this complex has the mobility of
S-labeled LBP. As
the amount of
H-labeled LPS used is increased, a high
mobility complex
H-labeled LPS and
S-labeled
LBP is formed ( panels c-e). However, the mobility of the
complex is not determined by the absolute amount of
H-labeled LPS used, but rather the
H-LPS:
S-LBP ratio, as is seen by comparing
panel f with d and e. In summary, these data
suggest that
H-labeled LPS and
S-labeled LBP
may form two sorts of complex depending on the LPS:LBP ratio. One
complex contains a single LBP and a small number
(1, 2) of LPS molecules. The other contains a large number of LPS
and probably multiple LBP.
Figure 1:
Sucrose
density gradients of mixtures of H-labeled [LPS and
S-labeled] LBP. Sedimentation direction is from right
to left.
H-Labeled LPS and
S-labeled LBP were
mixed at the concentrations shown in Table I for 15 min at 37 °C
before chilling to 4 °C for centrifugation. See text for
methods.
Sucrose Density Gradient Analysis of LPS
When sCD14
Complexes
H-labeled LPS,
S-labeled
sCD14, and LBP are mixed in various concentrations (see )
and subjected to sucrose density gradient sedimentation, an
H-LPS
S-sCD14 complex forms only in the
presence of LBP, as shown in Fig. 2. As was the case with
S-labeled LBP,
H-labeled LPS and
S-labeled sCD14 sediment distinctly differently in the
sucrose gradients (Fig. 2, panels a and b).
However, in contrast to the case with
S-labeled LBP, no
obvious
H-LPS
S-sCD14 complex is formed
when the two are simply mixed (Fig. 2, panel c).
Addition of a small amount of LBP does enable an
H-LPS
S-sCD14 complex to form
(Fig. 2, panel d). With
S-labeled sCD14,
however, only one size of
H-LPS
S-sCD14
complex is seen despite the use of relatively very high concentrations
of
H-labeled LPS (Fig. 2, panels e and
f) compared to the concentrations used to generate large
complexes of
H-labeled LPS with
S-labeled LBP.
The mole ratio of LPS:sCD14 in the complexes (see ) is
distinctly less than that in the incubation mixtures when LPS is in
excess. Both the size and composition of the LPS
sCD14 complexes
suggest that sCD14 can only complex with a limited number of LPS
molecules.
Figure 2:
Sucrose density gradients of mixtures of
H-labeled LPS,
S-labeled sCD14, and LBP.
Sedimentation direction is from right to left.
H-labeled
LPS,
S-labeled sCD14, and LBP were mixed at the
concentrations shown in Table II for 15 min at 37 °C before
chilling to 4 °C for centrifugation. See text for
methods.
Enhanced Emission from FITC-LPS Upon Binding to LBP or
sCD14
To develop a system that would permit real time
determinations of the kinetics and equilibria governing interaction of
LPS with LBP and sCD14 we asked whether the fluorescence signal of
FITC-LPS would change upon complexation with LBP or sCD14. We observed
an approximately 3-fold enhancement of fluorescence. The physical basis
for this enhancement is not totally defined, but probably derives from
relief of self-quenching in aggregates of FITC-LPS. S. minnesota Re595 LPS is undoubtedly strongly aggregated as are many types of
LPS
(14) . We observed that
n-octyl--D-glucopyranoside also enhances the
fluorescence, again suggesting that aggregate dissociation is
responsible. As indicated by the sucrose density gradients of Figs. 1
and 2, incubation of
H-labeled LPS with excess LBP or sCD14
reduces it's sedimentation velocity essentially to that of the
protein, suggesting disaggregation of the
H-labeled LPS.
Equilibria and Kinetics of FITC-LPS Binding to
LBP
The time dependence of the fluorescence change when 4.2
10
M FITC-LPS reacts with a 10-fold
excess of LBP (4.2
10
M) is shown
in Fig. 3 a. The initial fluorescence is that of the
buffer alone. FITC-LPS and LBP were added at points
`` a'' and `` b'' respectively. The
fluorescence change from 120 s, a time just beyond point b, to 250 s
was fit as a first order process. The individual data points and the
calculated first order curve are shown in the inset with
k
= 4.7
10
s
. Experiments like these were conducted at a
variety of FITC-LPS and LBP concentrations to characterize the kinetics
of FITC
LPS
LBP complex formation. Over 2 orders of magnitude
in [LBP], 4
10
to 4
10
M, k
did not
change significantly. These data suggest that formation of
FITC
LPS
LBP complexes from LPS
and LBP is a
two-step process as shown in Fig. S1and that the molecularity of
LBP does not change during the rate-limiting step. Because we can
directly observe LBP bound to LPS aggregates (see Fig. 1) we
favor a reaction scheme for FITC-LPS-LBP formation in which LBP rapidly
binds to FITC-LPS aggregates. Subsequently, FITC
LPS
LBP
complexes dissociate from these aggregates in the rate-limiting step.
Figure S1:
Scheme 1
The rate of FITC-LPS-LBP formation was found to be strongly
dependent on the salt concentration. The value of k was determined in 10 mM phosphate buffer at several
concentrations of KCl as shown in Fig. 3 b. This
observation is consistent with the idea that FITC-LPS-LBP formation
involves transfer of hydrophobic structures such as the fatty acid
tails of lipid A through a polar environment
(15) .
for FITC
LPS
LBP complexes
was obtained by analyzing the dependence on [LBP] of the
difference in fluorescence between FITC-LPS alone and at the end of the
association reaction. The data and their fit to an hyperbola are shown
in Fig. 3 c from which K
= 0.81(± 0.34)
10
M. The average of three determinations of
K
was 3.5
10
M with a range of 0.81 to 6.1
10
M.
Equilibria and Kinetics of FITC-LPS Binding to
sCD14
Because complexes of LPS with either mCD14 or sCD14 are
critical for activation of most LPS-sensitive cells, we investigated
the reaction of FITC-LPS with sCD14. The data of
Fig. 4
demonstrate the role of LBP in the binding of FITC-LPS to
sCD14. At time A, 10 ng/ml FITC-LPS (4.5
10
M) was added to the cuvette, followed by
10 µg/ml sCD14 (1.8
10
M) at
time B. The fluorescence does not change until time
C, when LBP is added at 0.04 µg/ml (6.6
10
M). At the end of the ensuing
fluorescence change, i.e. at about 300 s elapsed time, one may
ask whether LBP has limited the reaction, since it is at the lowest
concentration. Addition of another aliquot of LBP at C` does
not cause a further fluorescence change, nor does addition of another
aliquot of sCD14 at B`. Only addition of more FITC-LPS at
A` causes a further increase in fluorescence. Since LBP was
not limiting at the end of the first reaction, since almost no reaction
occurred in its absence, and because 7 mol of FITC-LPS/mol of LBP bound
to sCD14, we conclude that LBP must have catalyzed the binding of
FITC-LPS to sCD14. In the experiments summarized in
Fig. 5b, at the lowest LBP concentrations used nearly
500 mol of FITC-LPS bound to sCD14/mol of LBP.
Figure 4:
Fluorescence intensity versus time upon sequential addition of 4.5 10
M FITC-LPS at A and A`, 1.8
10
M sCD14 at B and B`,
and 6.7
10
M LBP at C and
C`. Note that these concentrations are in the relative
proportions of
LBP(1):FITC-LPS(6.7):sCD14(268).
Figure 5:
a, the net change in fluorescence upon
mixing sCD14 with 4 10
M FITC-LPS
in the presence of 5
10
M LBP. The
solid line is fit to a quadratic hyperbola with K apparent = 2.9
10
M.
See methods for details. b, the apparent first order rate
constant for FITC
LPS
sCD14 complex formation versus LBP concentration with [sCD14] = 4.5
10
M and [FITC-LPS] = 8
10
M. The solid line is
calculated for a rectangular hyperbola with k
= 8.9
10
s
and the [LBP] at which k
= 0.5
k
is 1.1
10
M. c, the apparent
first order rate constant for FITC
LPS
sCD14 complex
formation versus sCD14 concentration with [LBP]
= 4.5
10
M and
[FITC-LPS] = 8
10
M. The solid line is calculated for a
rectangular hyperbola with k
= 6.6
10
s
and the
[sCD14] at which k
= 0.5
k
is 2.5
10
M.
When the magnitude of
the fluorescence changes such as those shown in the first part of
Fig. 4
are measured as a function of sCD14 concentration, the
Kfor FITC
LPS
sCD14 complexes
can be determined to be 2.7
10
M as
shown in Fig. 5 a. From four similar experiments, the
K
determination averaged 2.9
10
M (± 30%). Changing the FITC-LPS
concentration 4-fold, and changing the LBP concentration 2-fold had no
observable effect on the value of the K
.
This is consistent with the sucrose density gradient data which suggest
that LBP is not a constituent of the FITC
LPS
sCD14
complexes.
LPS
sCD14
complexes as a function of LBP and sCD14 concentrations are shown in
Fig. 5
, b and c. In the absence of LBP a rate
constant of 1.7
10
s
was
observed. Fig. 5, b and c, suggest that as the
protein concentration increases, there is a change in the rate-limiting
step of the overall reaction, as discussed in the context of
Fig. S1
.
Competitive Binding of LBP and sCD14 to LPS
As
discussed above for Fig. 4, when LBP is at a low concentration
relative to sCD14, the FITC-LPS transfers to sCD14. However, the
determined values for the relative Kvalues of FITC-LPS-LBP and FITC-LPS-sCD14, 3.5
10
M and 29
10
M, respectively, suggest that as the ratio of LBP to
sCD14 changes, the association of FITC-LPS with LBP and sCD14 should
change. More specifically, at an LBP:sCD14 ratio of 1:8 the FITC-LPS
should be approximately equally distributed between the two proteins.
Since the fluorescence signals of the FITC
LPS
LBP and
FITC
LPS
sCD14 complexes are essentially identical and will
not permit verification of this prediction, we have used the
radioiodinated, photoactivatable derivative of Re595 LPS, namely
[
I]ASD-LPS, to verify this prediction.
Fig. 6
shows the relative labeling of LBP and sCD14 when 5
10
M (0.3 µg/ml) LBP and varying
amounts of sCD14 are incubated and photolyzed with 8
10
M [
I]ASD-LPS. As
can be seen the labeling of the two proteins is roughly equal at an
sCD14 concentration of between 2
10
and 5
10
M, verifiying the prediction
made from the values of K
determined with
FITC-LPS.
(
)
have observed
that the fluorescein in the FITC derivative of E. coli 0111:B4
LPS is probably more distantly removed from the lipid a moiety than is
the fluorescein in the FITC-LPS used here; thus self-quenching in that
derivative is probably less pronounced. Nevertheless, the opportunity
to make any real time, continuous measurement of even one LPS binding
to LBP and sCD14 has considerable value. The reactions between FITC-LPS
and other LPS binding proteins such as bactericidal/permeability
increasing protein, cholesterol ester transport protein, and Limulus
anti-lipopolysaccharide factor are currently under study and will be
described elsewhere.
I]ASD-LPS in the presence of any
concentration of sCD14, but, as shown in Fig. 6, it is not. And
if LBP is a component of the FITC
LPS
sCD14 complexes, the
concentration of LBP should alter the apparent binding constant
obtained when sCD14 is increased, but it does not. Thus three lines of
evidence argue that LBP is not a constituent of the final complexes.
Furthermore, Figs. 3 and 5 b show that LBP accelerates the
reaction and that it delivers anywhere from 7 to nearly 500 molecules
of FITC-LPS to sCD14. Thus LBP accelerates the reaction, LBP is not
consumed in the reaction, and it does not perturb the final
equilibrium. Thus we conclude that it is fair to claim that LBP acts as
a catalyst in the reactions described.
) with LBP are
summarized in in reactions 1-4 of Fig. S1. If the pathway
involving reactions 1 and 2 is operative, then LBP reacts with
FITC-LPS
followed by dissociation of complexes of LBP
with monomeric FITC-LPS. In this sequence, reaction 2 would be the
rate-limiting step since reaction 2 involves no change in the
molecularity of the reaction and the rate of LPS
LBP complex
formation was observed to be independent of the LBP concentration. If
the alternative pathway involving reactions 3 and 4 is operative,
monomers of FITC-LPS dissociate from the FITC-LPS
and
then react with LBP. In this sequence reaction 3 would be the
rate-limiting step. Between these two pathways for LPS-LBP formation,
we favor the upper pathway 1 and 2 primarily for the following reasons.
The first is that complexes of LBP with LPS aggregates are directly
observable in the sucrose density gradients. Complex formation via 3
and 4 does not involve any buildup of LBP bound to LPS aggregates since
reaction 3 is rate-limiting. Second, if LBP reacted only with LPS
monomers, because it has a higher affinity for LPS than does sCD14, LBP
should inhibit the reaction of FITC-LPS with sCD14, not catalyze it.
Undoubtedly there is some low equilibrium concentration of LPS monomers
(21) which could react directly either with LBP or sCD14.
Presumably it is this pathway which provides for the spontaneous
reaction of FITC-LPS with sCD14 with a rate constant of 1.7
10
s
.
from the data of both Fig. 5, b and c,
should be approximately 4.2
10
s
, i.e. the rate constant for
dissociation of an FITC
LPS
LBP complex from
FITC-LPS
-LBP. The observed values are 8.9
10
s
and 6.6
10
s
, respectively. Given the
scatter in the data and the other vagaries in these reactions which we
have experienced, we feel that theory and experiment are in acceptable
agreement. Thus we conclude that the pathway for the formation of
LPS
sCD14 complexes catalyzed by LBP is that which is shown in
boldface in Fig. S1.
LBP complexes
(16) .
Thus while LBP plays a catalytic role in the formation of
LPS
sCD14 complexes for binding to non-mCD14 bearing cells, LBP
does not have a strictly catalytic role in the presentation of LPS to
mCD14 bearing cells. Hailman et al.(7) have shown
that LPS
sCD14 complexes at 500 ng/ml LPS and 3 µg/ml sCD14
are agonists for neutrophils in the absence of LBP. However, Weingarten
et al.(23) have shown that 0.01 ng/ml LPS was a
direct agonist for neutrophils in whole blood. Thus the presence of LBP
can apparently make 4 orders of magnitude difference in the ability of
LPS to activate neutrophils. Similar effects are seen with monocytes
(24) and this may be related to association of LBP with the
cells.
for
FITC-LPS complexes of sCD14 and LBP; sCD14 binds FITC-LPS some 10-fold
less tightly than LBP.
Table: Fig. 1 sucrose density gradients: LPS
and LBP
Table: Fig. 2 sucrose density gradients: LPS,
sCD14, and LBP
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.