(Received for publication, August 2, 1995; and in revised form, December 11, 1995)
From the
Lipopolysaccharide (LPS) binding protein (LBP) is a lipid
transfer protein that catalyzes transfer of LPS monomers from micelles
to a binding site on soluble CD14 (sCD14) and transfer of LPS from
LPSsCD14 complexes to HDL particles. To characterize the first of
these two reactions, LPS covalently derivatized with the fluorophore,
boron dipyrromethene difluoride (BODIPY), was used to monitor
LBP-catalyzed movement of LPS in real time. The fluorescence efficiency
of micelles of BODIPY-LPS was low but was strongly increased upon
dissolution in detergent or upon binding to sCD14. Spontaneous binding
of BODIPY-LPS to sCD14 was very slow but was accelerated by
substoichiometric concentration of LBP, and the rate of binding was
measured under a variety of conditions. LBP-catalyzed transfer was
first order with respect to both sCD14 and LPS concentration, and the
apparent K
values were 1
2 µg/ml
for sCD14 and 100 ng/ml for LPS. The maximum turnover number for LBP
was approximately 150 molecules of LPS min
LBP
. LBP alone caused a small but measurable
increase in the fluorescence of BODIPY-LPS, suggesting that it bound
LPS aggregates but did not readily remove LPS monomers. The subsequent
addition of sCD14 caused a large fluorescence increase, suggesting
transfer of BODIPY-LPS to sCD14. These and other observations suggest
that LPS is transferred by an ordered ternary complex reaction
mechanism in which LBP transfers LPS monomer from LPS aggregates to
sCD14 without dissociating from the LPS aggregate.
Mammals mount an innate immune response to Gram-negative
bacteria by recognizing lipopolysaccharide (LPS, endotoxin), ()an amphipathic molecule that forms micelles in aqueous
buffers. Micelles of LPS bind poorly to leukocytes and provoke a
response only at very high concentrations. The addition of plasma,
however, dramatically enhances the ability of LPS to both bind to cells
and evoke responses(1, 2) . Two plasma proteins, LPS
binding protein (LBP) and soluble CD14 (sCD14) have been shown to play
a large role in transporting LPS and in mediating responses of
cells(3, 4) . LBP is a lipid transfer protein that
catalyzes transfer of LPS monomers from micelles to a binding site on
sCD14(5) , from LPS-sCD14 complexes to HDL particles (6) and from LPS micelle to HDL particle(7) . By
transferring LPS first to sCD14 and from LPS
sCD14 to HDL, LBP
effects transport of LPS(6) . As a first step in obtaining the
catalytic constants for the above three reactions, we have prepared a
fluorescent derivative of LPS that exhibits a large spectral shift upon
movement from a micelle to sCD14, and we have used this compound to
measure the turnover number for the LBP-catalyzed binding of LPS to
sCD14 as well as a K
for each reactant.
Our studies suggest that the transfer reaction proceeds via an ordered
ternary complex of LPS, LBP, and sCD14.
Figure 1: Emission spectrum of BODIPY-LPS. The fluorescence spectrum of 10 µg/ml BODIPY-LPS, with (B) or without (A) 2% SDS was recorded at room temperature with excitation at 485 nm. Note the different scales on the y axis.
Fluorescence intensity was
converted to molecules of LPS transferred using a BODIPY-LPS/SDS
standard curve relating fluorescence to the concentration of
BODIPY-LPS. These standards were prepared daily, and the diluent
contained 2% SDS to allow maximal fluorescence. Since the fluorescence
of the BODIPY-LPSsCD14 complex was only 0.41 times as great as
BODIPY-LPS in SDS (see below), the standard curve was calibrated
accordingly.
Dissolution of BODIPY-LPS micelles with SDS led to a pronounced change in the emission spectrum with a strong maximum at 518 nm (Fig. 1B). This observation further confirms the attachment of BODIPY to LPS and indicates that fluorescent properties report the aggregation state of BODIPY-LPS. Since the emission at 518 nm rises up to 50-fold upon disaggregation of BODIPY-LPS, we have used fluorescence intensity at this wavelength as a measure of disaggregation.
Figure 2: Fluorescence change caused by LBP/BODIPY-LPS interaction. 16.5 nM (50 ng/ml) BODIPY-LPS was mixed with increasing concentrations of LBP in PD buffer at 37 °C. The fluorescence at 518 nm rose rapidly (not shown) and was measured at the plateau (about 20 s after the addition of LBP). The data are the mean of three fluorescence measurements ± S.D. of a representative experiment repeated three times. Fluorescence of 16.5 nM BODIPY-LPS in 2% SDS was 0.552.
Figure 3: LBP-mediated transfer of BODIPY-LPS to sCD14. 13.2 nM BODIPY-LPS, 0.67 nM LBP and 90 nM sCD14 in different combinations were mixed at 30 °C as indicated in the figure, and the fluorescence at 518 nm was recorded over time. The figure is representative of three separate studies.
Figure 4: Fluorescence at plateau of BODIPY-LPS depends on the concentration of sCD14. 330 nM BODIPY-LPS, 16.7 nM LBP, and increasing concentrations of sCD14 were mixed and incubated at room temperature for 4 min prior to measuring fluorescence emmision at 518 nm (open square, right axis). Fluorescence measurements for BODIPY-LPS and sCD14 mixtures without LBP are shown with open circles, right axis. The concentration of BODIPY-LPS in BODIPY-LPS/sCD14 complexes was calculated from the fluorescence measurement as described under ``Materials and Methods'' and is shown on the left axis. The fluorescence of 330 nM BODIPY-LPS rose to 1.3 with 2% SDS. The data are the mean of two fluorescence measurements ± S.D. of a representative experiment repeated three times.
To obtain a preparation of BODIPY-LPS/complexes that
did not contain any free BODIPY-LPS micelles, 200 ng/ml BODIPY-LPS was
incubated with 1 µg/ml LBP and a 15-fold molar excess of sCD14 (50
µg/ml) for longer than 300 s to achieve maximal transfer. In the
presence of sCD14 and LBP, the fluorescence intensity at 518 nm rose
10.5-fold (from 0.031 to 0.322). The addition of SDS to this mixture
caused the fluorescence to rise a further 2-fold, confirming that sCD14
had not fully increased the fluorescence efficiency. The mixture was
then passed through an ultrafiltration membrane with a 100-kDa size
cutoff (see ``Materials and Methods''). Previous studies
showed that both sCD14 and LPS
sCD14 complexes pass through the
membrane, but neither LPS micelles alone nor LBP pass this
filter(6) . We verified that less than 2.5% of BODIPY-LPS alone
passed through the filter (not shown). The ultrafiltrate showed strong
fluorescence at 518 nm with an emission spectrum identical to that of
BODIPY-LPS in SDS (not shown). This observation confirms that
BODIPY-LPS
sCD14 complexes are formed and that they show the
fluorescence properties of disaggregated fluorophore. The addition of
SDS directly to the ultrafiltered complexes caused the fluorescence to
rise. After correction for background, the fluorescence of
BODIPY-LPS
sCD14 complexes was 41.3 ± 2.3% (n = 3) of BODIPY-LPS in 2% SDS. This observation indicates
that BODIPY-LPS in complex with sCD14 shows lower fluorescence
efficiency than BODIPY-LPS in SDS, and we have used this finding to
calculate the concentration of BODIPY-LPS
sCD14 complexes. On the
day of each experiment, we measured the fluorescence of BODIPY-LPS
alone and in the presence of 2% SDS. A curve relating fluorescence to
BODIPY-LPS
sCD14 concentration was then plotted with 0% as the
fluorescence value of BODIPY-LPS alone and 100% as the value of
BODIPY-LPS measured in SDS
0.41. We have used this method to
determine the amount of BODIPY-LPS bound to sCD14 in the presence of
excess BODIPY-LPS (Fig. 4, left y axis). At several
sCD14 concentrations, a constant proportion of 1 BODIPY-LPS appeared
bound per sCD14.
To verify that the movement of BODIPY-LPS
into sCD14 was caused by LBP rather than a contaminant or buffer
constituent, anti-LBP IgG was added to the reaction. Rabbit IgG had
little effect on movement of BODIPY-LPS to sCD14, but anti-LBP
completely blocked transfer (Fig. 5). The inhibitory effect of
antibody could be seen at an antibody concentration as low as 1
µg/ml. To verify that the LPS binding site on sCD14 is required for
the LBP-catalyzed rise in fluorescence, we employed a mutant sCD14 in
which residues 57-64 of the LPS binding site are deleted
(sCD14). This mutant sCD14 shows strongly
decreased ability to bind LPS in gel assays or to mediate cellular
responses to LPS(9) , and we found that
sCD14
showed a strongly decreased ability to
enhance fluorescence of BODIPY-LPS under the same experimental
conditions (Fig. 6).
Figure 5: Anti-LBP antibody blocks transfer activity of LBP. Anti-LBP (line B) or nonimmune rabbit IgG (line A) (20 µg/ml) was incubated with LBP (40 ng/ml) for 5 min at room temperature. BODIPY-LPS (50 ng/ml) and sCD14 (2 µg/ml) were then added, and the fluorescence of the mixtures was recorded over time at 30 °C. The other lines are the mixtures as indicated in the graph. The experiment is representative of three repeats.
Figure 6:
sCD14 fails to
enhance the fluorescence of BODIPY-LPS. 13.2 nM BODIPY-LPS and
0.67 nM LBP were mixed with either 5 µg/ml full-length
sCD14 (line A) or sCD14
(line
B), and fluorescence at 518 nm was recorded over time at 30
°C. Control mixtures were as shown on the graph. The figure is representative of two separate
experiments.
In the above studies, sCD14 was present in stoichiometric excess of LBP. In blood, however, approximately equal levels of LBP and sCD14 are present, and Tobias et al.(15) have suggested that under these conditions the LPS will remain with LBP and will not be transferred to sCD14. We tested this hypothesis by measuring the interaction of BODIPY-LPS with equimolar LBP and sCD14 (Fig. 7). LBP alone, at a concentration stoichiometric to LPS (line B), caused some enhancement of the fluorescence of BODIPY-LPS. When stoichiometric sCD14 was added to the mixture of LBP and BODIPY-LPS the fluorescence increased very rapidly (line D), suggesting rapid transfer to sCD14. The level of fluorescence obtained was similar to that seen with substoichiometric LBP (line E). This result suggests that BODIPY-LPS is substantially transferred to sCD14 even in the presence of stoichiometric levels of LBP.
Figure 7: BODIPY-LPS is transferred to sCD14 in the presence of equimolar LBP. In lines A-C, BODIPY-LPS (33 nM), LBP (33 nM), and sCD14 (36 nM) were mixed as shown, and fluorescence was recorded for 500 s at 30 °C. In line D, BODIPY-LPS and LBP were mixed and measured for 200 s, sCD14 was then added, and measurement was continued. In line E, 10-fold less LBP (3.3 nM) was employed. Results are representative of three repeats.
Figure 8:
Dependence of initial transfer rate on the
concentration of sCD14 and BODIPY-LPS. The initial rate (V) of BODIPY-LPS transfer to sCD14
catalyzed by 40 ng/ml LBP was measured at 30 °C in PBS. A,
the concentration of sCD14 was fixed at 1, 2, or 5 µg/ml, and
increasing amounts of BODIPY-LPS were added. Inset, a
secondary plot of slope versus 1/CD14. B, the
concentration of BODIPY-LPS was fixed at 30 or 60 ng/ml, and increasing
amounts of sCD14 were added. Data were analyzed by the Lineweaver-Burk
transform. Each data point is the average of three measurements. Graphs A and B are one of four experiments (two in
PBS, two in PD) with similar results. The slopes in both graph A (between 1 and 5 µg/ml CD14) and graph B (between 30
and 60 ng/ml LPS) were significantly different by the paired t test (n = 2, p =
0.05).
Here we describe a sensitive method for measuring the
movement of labeled LPS from micelles to sCD14. The method depends on a
spectral shift in a fluorophore upon dilution and is similar in
principal to a method recently described by Tobias et
al.(15) . Dilution of our BODIPY-LPS yields up to a
50-fold rise in fluorescence. Like Tobias et al., we confirm
the finding of Hailman et al.(5) that LBP catalyzes
transfer of LPS from micelles to sCD14 and that sCD14 binds
approximately one LPS molecule. We further show that the transfer
exhibits first order kinetics, and we have defined the catalytic
constants for the transfer reaction. We find that the K for sCD14 is 1-2 µg/ml, values within the range of
concentrations of sCD14 in human plasma. The K
for
LPS (
100 ng/ml), on the other hand, is substantially higher than
the LPS levels normally observed in sepsis, and LBP may thus normally
operate with levels of this substrate well below its K
. With saturating levels of LPS and sCD14, the
turnover number for LBP is approximately 150 mol of LPS
min
LBP
.
Figure 9: Models for reaction mechanism of LBP. A, binary complex model. B, ternary complex model. See ``Discussion'' for details.
Figure 10:
Three transfer reactions facilitated by
LBP. LBP catalyzes movement of LPS monomer from LPS aggregates to
sCD14(5) , from LPS aggregates to HDL particles(7) ,
and from LPSsCD14 complexes to HDL
particles(6) .
Our conclusion that LBP transfers LPS via an ordered, ternary complex reaction model differs from that of Tobias et al.(15) , who favor the binary reaction model of Fig. 9A. These authors did not address the possibility of the ternary complex reaction of Fig. 9B. They argued that, since LBP binds to LPS with high apparent affinity, transfer to sCD14 would be energetically unfavorable. Our results indicate that, even with equimolar LBP and sCD14, transfer of LPS to sCD14 is favored (Fig. 7). How could LBP transfer LPS to sCD14 if its affinity for LPS is greater than that of CD14? We suggest that the high affinity of LBP is for the LPS aggregate, not the LPS monomer. A key feature of the ternary complex reaction model is that LBP may transfer an LPS monomer from an aggregate without the energetically unfavorable step of disassociating from the aggregate. One LBP molecule may thus transfer successive LPS monomers from a single aggregate (see curved arrow in Fig. 9B). This formulation is consistent with all known observations on the action of LBP.
While our results strongly suggest an ordered ternary complex mechanism for LBP-mediated transfer of LPS from micelles to sCD14, they do not describe the reaction mechanism for the other two reactions catalyzed by LBP: transfer of LPS from micelles to HDL particles and transfer of LPS monomers from sCD14 to HDL particles (see Fig. 10). Experiments to describe these reactions are currently under way.