(Received for publication, November 29, 1994; and in revised form, December 22, 1994)
From the
CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide
(LPS) and enables LPS-dependent responses in a variety of cells. Recent
limited proteolysis studies have implicated a region in CD14 between
amino acids 57 and 64 as being involved in LPS interaction. To
specifically assess the importance of this region with respect to LPS
binding, we constructed a mutant sCD14
(sCD14) lacking amino acids 57-64.
sCD14
was isolated from the serum-free
conditioned medium of this cell line, and, in all assays, the purified
protein failed to recognize LPS or enable LPS-dependent responses in
cells. We also demonstrated that the region between amino acids 57 and
64 is required for binding of a neutralizing CD14 mAb, MEM-18. Native
polyacrylamide gel electrophoresis assays were used to demonstrate that
MEM-18 and LPS compete for the same binding site on CD14. These data
strongly suggest that the region spanning amino acids 57-64 binds
LPS and that formation of sCD14
LPS complex is required in order
for sCD14-mediated responses to occur.
CD14 is a 55-kDa protein which exists as a
glycosylphosphatidylinositol-anchored protein found on the surface of
leukocytes or as a soluble protein found in
serum(1, 2, 3, 4) . CD14 binds one
molecule of lipopolysaccharide (LPS) ()in a reaction
catalyzed by LPS-binding protein (LBP(5) ), an acute phase
serum protein(6) . Complexes of LPS with CD14 have been shown
to be sufficient to elicit inflammatory responses in
leukocytes(5, 7, 8) and endothelial
cells(9, 10, 11, 12) . Neutralizing
mAbs to CD14 antagonize cellular responses to LPS in
vitro(1, 5, 9, 12, 13, 14, 15) ,
and recent experiments have shown that CD14 mAbs are also effective in vivo(23) . These observations suggest that CD14 may
be an important pharmacologic target for diseases mediated by LPS.
In order to better understand how CD14 enables cells to respond to LPS, we have initiated a series of studies aimed at characterizing the interaction between LPS and CD14. To date, we have shown that the amino-terminal 152 amino acids of CD14 binds LPS and enables normal cellular responses to LPS(8) . In our accompanying paper(16) , we demonstrate that LPS protects a region spanning amino acids 57-64 from proteolytic digestion without altering the overall conformation of CD14 (16) . These experiments suggested that amino acids 57-64 were involved in binding LPS.
In this
report we analyzed the requirement of amino acids 57-64 for LPS
binding by generating a mutant sCD14 protein
(sCD14) lacking this region. Here, we show
that purified sCD14
no longer binds LPS nor
activates cells. In addition, we mapped the epitope of a neutralizing
mAb, MEM-18, to the domain specified by amino acids 57-64 and
showed that MEM-18 and LPS compete for binding to this site on sCD14.
The Transformer site-directed mutagenesis
kit was also used to generate mutant cDNAs encoding sCD14 having
alanine substituted at various positions between amino acids 59 and 65.
For these experiments, the following mutant primers were used:
5`-GATGCGGACGCCGCCCCTAGGCAGTATGCTGACACG-3` for sCD14,
5`-GATGCGGACGCCGACGCGCGGCAGTATGCTGAC-3` for sCD14
,
5`-GCGGACGCCGACCCTGCGCAGTATGCTGACAC-3` for sCD14
,
5`-GACGCCGACCCGCGAGCGTATGCTGACACGGTC-3` for sCD14
,
5`-CGCCGACCCGCGTCAGGCTGCTGACACGGTTCAAG-3` for sCD14
,
5`-CCGCGGCAGTATGCTGCCACGGTCAAGGCTCTCC-3` for sCD14
, and
5`-GTCGATGCGGACGCCGCCGCGGCGGCGGCTGCTGCCACGGTCAAGGCTCTCCGC-3` for
sCD14
. Introduction of the appropriate
mutation in all cDNAs was confirmed by DNA sequencing.
To assess LPS binding of purified sCD14 preparations,
sCD14 or sCD14
were
incubated at various concentrations (0, 101, 303, and 909 nM)
with 3 µg/ml [
H]LPS prepared from E. coli K12 strain LCD25 (List Biological Laboratories) in the presence or
absence of 16.7 nM rLBP. The reaction was incubated at 37
°C for 30 min and then electrophoresed on native 4-20%
polyacrylamide gels. Gels were prepared for fluorography as described
previously(5) .
Experiments were also performed to determine
whether various CD14 mAbs could compete with LPS for binding to sCD14.
In these studies, [H]LPS
sCD14 complexes
were formed by incubating 130 µg/ml sCD14
with 10 µg/ml [
H]LPS for 15 h at 37
°C in PBS with 1 mM EDTA. Complexes were then diluted
10-fold and incubated for 20 min at 37 °C with 200 µg/ml
concentrations of various mAbs in a total volume of 10 µl. Mixtures
were then electrophoresed on 8-16% native gels and processed for
fluorography as above.
In other experiments, we examined whether
rLBP could lower the effective dose of LPS required to competitively
inhibit binding of MEM-18 or 3C10 to sCD14. MEM-18 or 3C10 (40
µg/ml) was incubated with sCD14 (2.6
µg/ml) for 10 min at 37 °C. Various concentrations of LPS (from S. minnesota strain R60, List Biological Laboratories) were
then added in the presence or absence of rLBP (1 µg/ml) for 20 min
at 37 °C in a total volume of 10 µl. Mixtures were then
electrophoresed on 8-16% native gels and transferred to
nitrocellulose in Tris-glycine buffer with 20% methanol. The
nitrocellulose was blocked in PBS with 10% dry milk and incubated with
polyclonal antibodies in PBS with 0.1% dry milk. CD14 was detected
using a rabbit polyclonal antibody (generous gift of Dr. Pat Detmers)
raised against sCD14
and an alkaline
phosphatase-conjugated secondary antibody. Bound antibody was detected
using p-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate-toluidine salt according to the
manufacturer's instruction.
We then used a native PAGE assay (5, 8) to assess
whether sCD14 present in COS-7 CM binds LPS.
CM containing sCD14
or sCD14
were incubated with increasing amounts of LPS, and the mixtures
were electrophoretically transferred to nitrocellulose membranes. sCD14
or sCD14
LPS complexes were then detected with anti-CD14
polyclonal antiserum. Fig. 1shows that LPS caused a shift in
the electrophoretic mobility of sCD14
, and
previous studies showed that this shift is caused by binding of LPS to
CD14(5) . In contrast, no shift was observed in CM containing
sCD14
even at an LPS concentration 5-fold
higher then that needed to completely shift
sCD14
. These results are consistent with the
notion that amino acids 57-64 in sCD14 are critical for LPS
binding.
Figure 1:
COS-7 CM containing
sCD14 does not form complexes with LPS.
Thirty µl of CM containing sCD14
or
sCD14
were incubated with 1 µg (lanes 2 and 6), 2 µg (lanes 3 and 7), or 5 µg (lanes 4 and 8) of Re 595
LPS for 30 min at 37 °C. Lanes 1 and 5 represent
CM alone without LPS. Protein mixtures were run on 10% native
polyacrylamide gels and then transferred to nitrocellulose membranes.
sCD14
was detected using polyclonal anti-human
CD14 antiserum.The position of sCD14
alone or
LPS
sCD14
complexes are indicated by arrows.
Figure 3:
sCD14 but not
sCD14
mediates responses of PMN to LPS and
LBP. Freshly isolated PMN were incubated with ``smooth'' LPS (E. coli 0111:B4, 30 ng/ml), rLBP (1 µg/ml), and the
indicated concentrations of sCD14
or
sCD14
for 10 min at 37 °C. Cells were
washed, and adhesion to fibrinogen-coated wells was
measured(5, 22) . Error bars indicate
standard deviations of triplicate
determinations.
Figure 2:
sCD14 does not
form stable complexes with [
H]LPS. Various
concentrations of sCD14
(lanes
2-4) or sCD14
(lanes
5-7) were incubated without rLBP (A) or with 16.7
nM rLBP (B) as described under ``Materials and
Methods.'' Lane 1 contains LPS in the absence of
additional protein. Mixtures were run on 4-20% native
polyacrylamide gels and processed for fluorography. Positions of
uncomplexed LPS, and complexes between LPS and sCD14
are indicated.
We also examined
the ability of sCD14 to support responses of
U373 cells to LPS. Addition of as little as 5 ng/ml
sCD14
enabled strong IL-6 production in response
to LPS (Fig. 4), confirming previous
reports(8, 9) . In contrast,
sCD14
failed to support LPS-dependent IL-6
production even at a concentration of 80 ng/ml. These findings confirm
that residues 57-64 are crucial to the biological function of
sCD14
.
Figure 4:
sCD14 does not
induce IL-6 production in U373 cells. U373 cells were treated with
various concentrations of sCD14
or
sCD14
in the presence or absence of LPS (10
ng/ml) for 24 h. IL-6 levels were determined as described(8) .
Data presented are means ± S.D. from four
readings.
Figure 5:
mAb MEM-18 does not recognize
sCD14. A, immobilization of mAb
3C10 to a sensor chip and injection of solutions at various
``Steps'' are detailed under ``Materials and
Methods.'' Wash indicates a washing step using HBS
buffer. Binding of purified proteins to mAbs was assessed by measuring
the change in response unit (RU) after a 2-min wash. B, various amounts (2 ng for lanes 1, 2, 3, and 5; 10 ng for lanes 4 and 6)
of purified sCD14
(lanes 1, 3,
and 4) or sCD14
(lanes 2, 5, and 6) were electrophoresed on 4-20%
SDS-PAGE, and proteins were transferred to nitrocellulose membranes.
For detection of sCD14 protein, polyclonal anti-CD14 antiserum (lanes 1 and 2) or mAb MEM-18 (lanes
3-6) were incubated with the filters for 1 h. Immune
complexes were detected by enhanced chemiluminescence (ECL, Amersham)
as described by the manufacturer.
In an attempt to further characterize the
MEM-18 epitope, we constructed a series of cDNAs encoding sCD14 having
alanine substituted at various positions between amino acids 59 and 65. Table 1summarizes the corresponding amino acid changes for each
mutant construct. Mammalian expression vectors containing each mutant
cDNA were transiently transfected into COS-7 cells, and expression of
mutant protein in CM was monitored by Western blot analysis. No
differences in expression of mutant sCD14 proteins were observed in
COS-7 CM (data not shown). Therefore, we performed BIAcore analyses to
test the ability of each CM containing mutant sCD14 to bind MEM-18.
Immobilized mAb 3C10 recognized each of the constructs, but
sCD14, sCD14
, sCD14
, and
sCD14
were not recognized by MEM-18 (Table 1). Binding of MEM-18 was not affected if Arg
or Asp
was mutated, and substitution of alanine at
Pro
partially inhibited MEM-18 binding. In summary, we
have demonstrated that MEM-18 recognizes an epitope which is minimally
comprised of residues Asp
, Gln
, and
Tyr
.
Figure 6:
LPS competes with mAb MEM-18 for binding
to sCD14. A, [H]LPS
sCD14 complexes
were formed as described under ``Materials and Methods.''
Complexes were then diluted 10-fold and incubated with buffer (lane
2) or mAbs MY4, 3C10, MEM-18, or IB4 (lanes 3-7,
respectively). The same concentration of [
H]LPS
was run in the absence of sCD14
for comparison (lane 1). Mixtures were run on 8-16% gels and processed
for fluorography. Free [
H]LPS,
[
H]LPS
sCD14, and
[
H]LPS
sCD14
mAb complexes are
indicated. B, purified sCD14
was
incubated with mAb MEM-18 (lanes 3-11) or 3C10 (lanes 12-13) in the absence of rLBP (lanes
3-7 and 12) or in the presence of rLBP (lanes
8-11 and 13) at a concentration of 16.7
nM. Increasing concentrations of LPS (0.25 µg/ml for lanes 4 and 8; 1 µg/ml for lanes 5 and 9; 5 µg/ml for lanes 6 and 10; 25
µg/ml for lanes 7, 11, and 13) were used
to competitively inhibit binding of MEM-18 to
sCD14
. MEM-18 (lane 2) and 3C10 (lane 14) were run without sCD14
to
demonstrate the specificity of the antibody used for blotting. Protein
mixtures were run on 8-16% native gels and processed for Western
blot analysis as indicated under ``Materials and Methods.''
Complexes of sCD14
LPS and
sCD14
mAb are
indicated.
To confirm and extend this
observation, complexes of MEM-18 and sCD14 were
first formed. These complexes showed a mobility characteristic of a
250-kDa protein, confirming the efficacy of MEM-18 in the supershift
assay (Fig. 6B, lane 3). Addition of
increasing doses of LPS to these complexes caused dissociation of the
sCD14
from the MEM-18 in a dose-dependent
fashion. Moreover, the efficacy of LPS in disassociating
sCD14
from MEM-18 was enhanced by rLBP, a protein
that catalytically hastens the binding of LPS to
sCD14
(5) . LPS did not cause
disassociation of sCD14
from 3C10 (Fig. 6B), MY4, or 60b (data not shown), confirming
that these mAbs do not compete with LPS for binding to
sCD14
. These results further confirm that MEM-18
and LPS bind sCD14
in a competitive fashion and
may thus recognize overlapping sites.
In this report, we provide compelling evidence that the region between amino acids 57 and 64 of sCD14 is essential for proper binding of LPS. Deletion of this region abolished the ability of sCD14 to bind LPS in the presence or absence of rLBP. Furthermore, an epitope recognized by neutralizing mAb MEM-18 was mapped to this region, and we showed that this mAb competes with LPS for binding to sCD14. These data are consistent with our previous finding (8) that localized an LPS binding site to the amino-terminal 152 amino acids of sCD14 and our accompanying paper (16) which demonstrates that LPS protects a region spanning amino acids 57-64 from cleavage by endoproteinase Asp-N protease.
Recently, an LPS binding motif has been proposed for LALF(18) . Identification of the putative binding site was based on the observation that in the crystal structure of LALF, there is an extended amphipathic loop which has sequence similarity to polymixin B, an antibiotic that binds lipid A. We observed that the region of highest amphipathicity in CD14 overlapped our proposed LPS binding domain. Interestingly, the amphipathic domain in CD14 has a net negative charge, distinguishing it from analogous domains in LALF, LBP, and BPI. These data support the theory that amphipathic domains are involved in LPS binding and also imply that amphipathicity may be more critical to LPS binding than net charge. However, more data are required to confirm whether amphipathic domains in LALF, LBP, and BPI truly bind LPS.
Our data also demonstrate the biological
consequences of impairing LPS binding to sCD14.
sCD14 was inactive in enabling PMN and U373
responses to LPS. These results suggest that binding of LPS to sCD14 is
a prerequisite for the biological activity of CD14. This conclusion is
consistent with the finding (5) that binding of LPS to
sCD14
is temporally correlated with biological
activity.
While the above studies have identified a region of
sCD14 involved in one function of
sCD14
(binding of LPS), additional sites may also
play important roles. rLBP catalyzes movement of LPS into
sCD14
, and recognition of sCD14
by rLBP may involve a separate site.
sCD14
LPS complexes interact with cells and
with high density lipoprotein particles, (
)suggesting yet
another site. Experiments to map these sites are currently in progress.