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*
Bristol-Myers Squibb Pharmaceutical Research Institute, Lawrenceville, NJ 08543;
Immunex Corp., Seattle, WA 98101; and Departments of
Psychiatry and Behavioral Sciences and
§
Periodontics, University of Washington, Seattle, WA 98195
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently the LPS binding protein (LBP)2/CD14 activation pathway (1, 2) has been described as a mechanism of microbial activation of the host inflammatory response. Although inflammatory responses to a wide variety of different microbial components are mediated through this pathway (1, 3, 4, 5, 6, 7, 8, 9, 10), little is known concerning how the host recognizes different microbial structures or how it regulates the intensity of the inflammatory response. Studies with LPS suggest that recognition of specific lipid A structural details most likely occurs after CD14 binding (11, 12). Kitchens and Munford (11) by careful titration of LBP and LPS have shown that deacylated LPS inhibits wild-type LPS activation at an uncharacterized site after CD14 binding in the LPS recognition pathway. Further, Delude et al. (12) conclusively demonstrated that inhibition of LPS responses by Rhodobacter sphaeroides lipid A and lipid IVA was not due to LPS recognition by CD14. These data are consistent with a proposal by Wright (13) that CD14 serves as a mechanism to concentrate microbial ligands at the host cell surface.
Although CD14 binds and shuttles a wide variety of different microbial
ligands, the mechanism of how this is accomplished is not clear.
Initial studies with truncation mutants identified the amino-terminal
region and three leucine repeat domains as sufficient for
Escherichia coli LPS binding and host cell activation.
Separate studies demonstrated that four CD14 deletion mutation proteins
representing removal of the most hydrophilic regions (labeled 1–4 in
Fig. 1
) were independently necessary for LPS ligand binding.
Subsequently, protease protection experiments, an additional deletion
CD14 mutation protein, and mAb epitope mapping identified region 4 as
required for E. coli LPS binding (14, 15).
Another study confirmed that region 3 is involved in E. coli
LPS binding with the use of multiple alanine replacement mutations and
monoclonal epitope mapping (16). Additional mutations and
monoclonal epitope mapping indicated that region 1 may be involved in
LPS signaling, but not binding (17). Work from our
laboratory, initially employing a peptide approach and then
incorporating single amino acid replacements, identified a residue not
located in any of these regions to be involved in LPS binding (see Fig. 1) (18). These data indicate that there are several
different regions located throughout the amino-terminal region of CD14
that could be involved in LPS binding. However, little is known about
the potential tertiary structure of this region, since there are no
known sequence similarities with other protein domains, nor has an
x-ray crystallographic structure been reported. Therefore, the
molecular mechanism employed by CD14 to bind a wide variety of
different microbial ligands remains elusive.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagent grade chemicals were obtained from Sigma (St. Louis, MO). HUVEC growth medium consisted of medium 199 (Life Technologies, Gaithersburg, MD0 containing 4 mM L-glutamine, 90 µg/ml heparin, 1 mM sodium pyruvate, 30 µg/ml endothelial cell growth stimulant (Biomedical Products, Bedford, MA), and 20% FBS (HyClone, Logan, UT). Medium 199 stimulation medium consisted of medium 199 containing 4 mM L-glutamine, 90 µg/ml heparin, 1 mM sodium pyruvate, 1 mg/ml human serum albumin, and 5% pooled normal human serum (Gemini Bioproducts, Calabasas, CA). RPMI 1640 medium was obtained from Life Technologies (Grand Island, NY). IL-8 levels in supernatants were measured using Cytoscreen IL-8 Immunoassay Kit (BioSource, Camarillo, CA). Pooled human sera were obtained from Gemini Bioproducts and were depleted of sCD14 as previously described (20). Protein A-Sepharose was obtained from Repligen (Cambridge, MA). HUVEC were obtained from Clonetics (San Diego, CA) and maintained in HUVEC growth medium. Cells were used at the fourth passage for all experiments described in this report.
Bacterial strains and purification of LPS
Helicobacter pylori ATCC 43504 was obtained from the American Type Culture Collection (Manassas, VA). Porphyromonas gingivalis ATCC 33277 was provided by Aaron Weinberg, Department of Periodontics, University of Washington (Seattle, WA). E. coli BMS A016 (serogroup O6) is a human blood culture isolate provided by Marie Coyle, Harborview Medical Center (Seattle, WA). Strains were examined for purity, properly identified, and stored at -70°C. Cultures were made from frozen bacterial stocks to avoid repetitive subculture. Bacterial cells were grown for LPS isolation as follows. P. gingivalis was grown anaerobically at 37°C for 2–3 days in large batch cultures by Lee Laboratories (Grayson, GA) using enriched Trypticase soy broth (Becton Dickinson, Cockeysville, MD) and was provided as frozen cell pellets. H. pylori ATCC 43504 was grown for 3–4 days on H. pylori agar medium under microaerophilic conditions at 37°C. E. coli was grown overnight in Trypticase soy broth at 37°C with shaking in room air.
Purified LPS from P. gingivalis ATCC 33277 and H. pylori ATCC 43504 were prepared by the cold MgCl2-ethanol procedure (21). Purified LPS from E. coli A016 was obtained by the phenol-water method (22). All LPS preparations were suspended in distilled water and were found to be free from contaminating nucleic acid and protein by measuring OD at 280 and 260 nm (OD280 and OD260). Gas chromatographic analysis for sugar and fatty acid composition revealed that the compositions of these purified LPS agreed with published data (23, 24, 25) and allowed calculation of average m.w. The m.w. for E. coli, P. gingivalis, and H. pylori LPS were estimated to be 9170, 7042, and 5405, respectively.
Construction and expression of sCD14:Rg
A cDNA fragment encoding the extracellular domain of wild-type CD14 was subcloned upstream of a cDNA fragment encoding the H, CH2, and CH3 domains of human IgG1 in the mammalian expression vector CDM7B as previously described (20). Similarly produced CD40 recombinant globulin (CD40:Rg) was used as a control.
Oligonucleotide-directed mutagenesis
Mutations were introduced into CD14:Rg by a two-step PCR
procedure (18). Overlapping PCR fragments were first
generated in separate reactions and then reannealed and extended in a
second PCR. In the first step, oligonucleotides (complement and reverse
complement) containing the desired codon changes flanked by 18
nucleotides on each side were synthesized for each mutation. Two
fragments were then synthesized by PCR using T7 (in pcDNA3) and the
reverse complement oligonucleotide to generate the 5' fragment and
CD14R5 (5'-CAAGTCCTGTGGCTTCCA GAG-3') and the complementary
oligonucleotide to generate the 3' fragment. After gel purification,
10 ng of each fragment was mixed and used as template in a second
PCR reaction using T7 and CD14R5 oligonucleotides. The resulting
480-nucleotide PCR fragment was gel purified, digested with
HindIII and XhoI, and used to replace the 5'
HindIII/XhoI fragment from the wild-type
CD14:Rg/pcDNA3 plasmid. All nucleotide changes were confirmed by DNA
sequencing. The fusion proteins were produced by transient expression
in COS cells and were purified from the spent COS cell supernatants
using protein A-Sepharose chromatography. Fusion protein stock
solutions were found to be free of endotoxin by the Limulus
lysate assay obtained from BioWhittaker (Walkersville, MD).
Generation of mouse mAbs
mAbs to human sCD14 were cloned by standard methods after fusion of a murine myeloma line with spleen cells from mice immunized with purified sCD14:Rg as previously described (20). Throughout the cloning procedure, culture supernatants were assayed for Abs using the ELISA method, with purified sCD14:Rg as the solid phase Ag. Select mAbs of the appropriate specificity were produced in high titer culture supernatants. The Abs were purified from the culture supernatants by protein A chromatography, extensively dialyzed into PBS, filter-sterilized, and stored at 4°C. The purified mouse anti-human sCD14 mAbs used in these experiments were 2G7 and 6C8. A commercial mouse anti-human CD14 mAb (MY4) was obtained from Coulter Immunology (Hialeah, FL).
The mouse anti-LPS mAbs used for detection of LPS binding to sCD14:Rg were 3B4 (IgM; anti-E. coli A016), 5B9 (IgM; anti-P. gingivalis ATCC 33277), and 7A9 (IgG1 anti-H. pylori ATCC 43504). They were cloned as described above using spleen cells from mice immunized with purified LPS.
ELISA for detection of LPS binding to immobilized sCD14:Rg
LPS binding to wild-type and mutations of sCD14 were measured in a capture ELISA format (20). Briefly, Immulon II (Dynatech, Chantilly, VA) 96-well plates were coated overnight at 4°C with 0.05 µg/well SCD14 wild-type or point mutation protein. The next day, the wells were emptied, and nonspecific binding sites were blocked with 100 µl/well of 0.5% BSA in PBS for 30 min at 37°C. The plates were then washed three times with PBS. Fifty microliters of LPS diluted in 1% CD14-depleted serum (source of LBP) was added per well, and the plates were incubated at 37°C for1 h. Unbound LPS was removed with four PBS washes, and bound LPS was detected with specific mouse anti-LPS mAb at 0.5 µg/ml in 0.5% BSA in PBS as previously described (20).
ELISA for detection of anti-CD14 mAb binding to immobilized sCD14:Rg
Immunlon II plates were coated with 0.05 µg/well of sCD14 wild-type or point mutation fusion proteins as previously described (18), and the amount of fusion protein that was captured was monitored with goat anti-human IgG, which reacts with the CD14:Rg fusion protein. mAbs MY4, 2G7, and 6C8 (0.02 µg/ml) were added, and ELISA detection was performed as previously described (20) with goat anti-mouse IgG (Sigma).
IL-8 functional assay
Primary HUVEC cultivation and the IL-8 ELISA were performed as described previously (18, 26), except that 1% sCD14-depleted serum, prepared as previously described (20), instead of 5% complete serum was used in the medium 199 stimulation medium.
LPS blocking experiments
The ability of the CD14 mAbs to block LPs binding to CD14 was examined. CD14 was captured on a 96-well ELISA plate as previously described (27), and 30 min before the addition of E. coli LPS (100 ng/ml), anti-CD14 mAbs or a negative control Ab (anti-CD40) were added. E. coli LPS binding was detected as previously described (20), except that the anti-E. coli LPS mAb was biotinylated (used at 3 µg/ml), because the usual second-step goat anti-mouse would react with the mouse mAbs.
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Results |
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). Charged residues were chosen, since
the previous analysis had indicated that charge reversal mutations had
the greatest effect on LPS binding (18). The CD14 mutation
proteins were than examined for their ability to bind LPS
(20) obtained from three different species of bacteria
(E. coli, P. gingivalis, and H.
pylori) such that a broader range of potential LPS binding sites
could be identified as previously described (20). Initial analysis revealed three different groups of CD14 mutation proteins based upon their LPS binding characteristics
Initially a survey was performed with nearly all the new CD14
mutation proteins. Statistical analysis was not performed, nor were
additional assays conducted with all the mutation proteins. Rather,
this analysis was used to initially identify those new CD14
mutation proteins that may have an effect on LPS binding. Analysis
of the data revealed that three different patterns of LPS binding to
the CD14 mutation proteins were observed (Table I). In the majority of mutation
proteins (group 1) no effect on LPS binding was observed (26 of 36
examined). In this group, consistent with the initial, more limited
analysis, none of the serine replacement mutations reduced LPS binding
(a total of 19 were examined). The second group (group 2, indicated in
bold in Table I) consisted of those mutation proteins that demonstrated
a reduced binding with either P. gingivalis or H.
pylori, but not E. coli LPS (E4K, D9K, E11K, D12K,
R53E, D59K). All these were charge reversal mutations. Interestingly,
no mutation proteins examined demonstrated reduced E. coli,
yet had normal P. gingivalis or H. pylori LPS
binding. It is suspected that this may relate to the higher affinity
for CD14 displayed by E. coli LPS (20),
although this possibility has not been formally examined. The third
group (group 3, indicated by underlining in Table I) consisted of two
CD14 mutation proteins that displayed reduced binding to all three LPS
ligands examined (D10K and R14E).
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Additional, more comprehensive binding assays were performed with
representative CD14 mutation proteins from each group. These assays
confirmed the three different patterns of LPS binding to the CD14
mutation proteins mentioned above (see Table I and Fig. 2). The serine replacement mutation
proteins in group 1 did not significantly reduce the binding of any LPS
species (D10S, E11S, D12S, R14S, R53S, D57S, and D59S;
p > 0.1, not significant by two-sample t
test). The charge reversal mutations in group 3 reduced the binding to
all the LPS species examined to near background levels (D10K and R14E;
p < 0.05 or 0.001, significant or highly significant
compared with wild-type CD14 depending upon the species examined by
two-sample t test). However, similar to our previous results
(18), not all charge reversal mutations reduced LPS
binding (for example, D57K).
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The differential abilities of E. coli, P.
gingivalis, and H. pylori LPS to bind mutant CD14
proteins D12K and D59K were further investigated (Fig. 3). Concentration-dependent increases in
the binding of all three LPS species examined were observed with both
mutant CD14 proteins. Consistent with earlier analysis (Fig. 2),
E. coli LPS bound to D59K to nearly wild-type levels, and
that to D12K was slightly reduced (a significant reduction in E.
coli LPS binding to CD14 mutation protein D12K was only observed
at 30 ng/ml LPS). In contrast, P. gingivalis and H.
pylori LPS binding to CD14 mutation protein D12K was significantly
reduced at all LPS concentrations examined (p
> 0.05, by two-sample t test). Examination of LPS binding
to CD14 mutation protein D59K revealed that P. gingivalis
(p < 0.05, by two-sample t test),
but not E. coli or H. pylori, LPS was reduced
compared with wild-type CD14. These analyses are consistent with the
observation that charge reversal mutations in CD14 residues D12 and D59
have preferential effects on LPS ligand binding
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The ability of the CD14 single-point mutation proteins to activate
IL-8 secretion in HUVECs with E. coli LPS was examined. An
assay that employed CD14-depleted serum that was supplemented with the
different CD14 mutation proteins was used to examine the ability to
elicit IL-8 secretion from human endothelial cells (Fig. 4). Several mutation proteins were
examined that did not demonstrate significantly lower E.
coli LPS binding (E11S, D57K, D59S) and also did not display a
significant difference in IL-8 secretion. Three separate experiments
were performed, and these CD14 single-point mutation proteins did not
alter the amount of IL-8 secreted by human endothelial cells in
response to 30 ng/ml E. coli LPS by >20% that of wild-type
protein (data not shown). Fig. 4 presents the data obtained with those
mutant CD14 proteins that displayed significant reductions in either
E. coli LPS binding or the binding of P.
gingivalis or H. pylori (group 1, D10K and R14E; group
2, E11K, D12K, R53E, and D59K from Fig. 2). The effect of increasing
LPS concentrations was examined. Each CD14 mutant was able to elicit
IL-8 secretion; however, the amount of LPS necessary to facilitate
secretion varied significantly among the CD14 mutants. Mutants D10K,
D12K, and R14E that displayed the poorest ability to bind E.
coli LPS were also the poorest activators of IL-8 secretion.
Little or no IL-8 secretion occurred with these CD14 mutations proteins
at concentrations of LPS <3 ng/ml. In contrast, E11K and D59K, two
CD14 mutation proteins that did not display a significant reduction
in E. coli LPS binding (p > 0.7 and
p > 0.3, respectively), facilitated IL-8 secretion
similar to wild-type CD14 (Fig. 4). The relative ability of mutant CD14
proteins to bind E. coli LPS was similar to their respective
ability to facilitate E. coli LPS-mediated IL-8 secretion.
One exception to this pattern is that mutant D12K bound E.
coli LPS slightly better than R14E (Fig. 2); however, R14E was
slightly better at facilitating IL-8 secretion. Interestingly, this
mutation occurs in the hydrophilic region previously identified as
being involved in LPS signaling (17). However, the
differences in relative binding and activation were small between these
CD14 mutation proteins and do not represent a significant alteration in
the general pattern that the amount of LPS binding correlates to the
degree of cellular activation. The correlation of LPS binding to
activation is consistent with the idea that a major conformational
change that independently affected CD14 function did not occur in these
CD14 mutation proteins.
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Binding studies with the different CD14 mutation proteins
employing three different anti-CD14 mAbs were performed. The
generation of these Abs has been previously described
(27). These Abs blocked LPS binding (Fig. 5). The blocking of E. coli
LPS to CD14 is shown in Fig. 5; however, these Abs also blocked
P. gingivalis and H. pylori LPS binding (data not
shown). In fact an analysis of 35 master wells with LPS-blocking
activity failed to identify any Ab that displayed selective blocking of
these three different types of LPS (data not shown). In addition, these
Abs bound tertiary epitopes on CD14, as determined by their inability
to bind heat-denatured CD14 by ELISA (data not shown). The inability of
these Abs to bind denatured CD14 was further analyzed by immunoblot
analysis that demonstrated that these Abs did not bind to SDS and
heat-denatured CD14 after SDS-PAGE (data not shown). Further studies
(23 different anti-CD14 master wells that blocked LPS binding were
examined) failed to reveal an Ab that bound a heat-denatured (or
presumably linear) epitope by ELISA (data not shown).
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Each mAb displayed a different pattern of binding to the CD14
mutation proteins, demonstrating that each Ab bound a different epitope
(Table II). In addition, as expected, the
mAbs provided a measure of protein conformation independent of LPS
binding. For example, CD14 mutation proteins from group 1 (D10S, E11S,
D12S, D57K, and D59S), which demonstrated no observable phenotype with
respect to LPS binding or host cell activation, demonstrated reduced or
no binding to at least one of the mAbs. These data demonstrate that
although the CD14 mutation did not influence LPS binding, mAb binding
was affected. Conversely, the three CD14 mutation proteins from group 3
all bound at least one CD14 mAb (one of these, R14E, bound all the
mAbs; another, E37K, bound two of three of the Abs). These CD14
mutation proteins displayed a significant reduction with all the LPS
species examined, making it more difficult to rule out potential
conformation effects of these charge reversal mutations. However, based
upon the mAb data, the mutations did not induce conformational changes
that altered Ab binding. Collectively, these data clearly demonstrate
that reductions in mAb binding to CD14 mutation proteins occur
independently of their LPS binding properties.
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m Abs 2G7 and 6C8 represent the second pattern. mAb 2G7 failed to react
with CD14 mutation proteins located in hydrophilic region 1, similar to
MY4 (D9K, D10K, E11S, E11K, and D12K), although a slightly different
pattern was observed. In contrast to MY4, this mAb also failed to bind
CD14 mutation protein E37K, a CD14 residue located in hydrophilic
region 3 on the linear sequence (see Fig. 1). mAb 6C8 binding was also
reduced by mutations located in two different hydrophilic regions found
in the amino-terminal sequence of CD14. CD14 mutation proteins R53E,
D57K, D59S, and D59K, at or near region 4, and D10S, D10K, and R14E, at
or near region 1, all reduced or eliminated mAb 6C8 binding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Charge reversal mutations had the greatest effect on LPS binding and
resulted in the identification of CD14 mutation proteins that were
significantly reduced in their ability to bind at least one LPS species
(group 2: E11, D12, and R53, D59; group 3: D10 R14 from this study and
E28 and E37 from Ref. 18). These data now indicate that
residues located in highly hydrophilic regions 1, 3, 4, and E28 (near
region 2) contribute to the LPS binding site (Fig. 1). There are two
possibilities for the inability of LPS to bind to these different CD14
mutation proteins. The residue altered in the mutation protein is
located at or near an LPS binding site of the protein, and the reversal
in side group charge alters the binding site sufficiently to making it
incompatible for LPS binding. This would suggest that the LPS binding
site is tertiary in nature, incorporating residues located throughout
the amino-terminal portion of the molecule, as opposed to a linear
sequence located in one region. Alternatively, the different charge
reversal mutations may have induced a minor conformational change in a
distally located LPS binding region. Definitive resolution of this
issue most likely will require determining the protein structure after
crystallization.
However, independent evidence consistent with the idea that the CD14 LPS binding site is tertiary in nature and is comprised of residues located throughout the amino-terminal region was obtained. mAb epitope analyses revealed that hydrophilic regions 1, 3, and 4 are located in close enough physical proximity so that residues in each region influence mAb binding.
A major component of the epitope for mAb 2G7 is found in highly
hydrophilic region 1 of CD14 (residues D9 to D12, Table II). Both group
2 and 3 LPS binding CD14 mutation proteins are found in this cluster of
charged residues. There is good evidence that residue E11, located in
this cluster, is a contact residue for Ab 2G7. The replacement of the
side chain carboxyl group of E11 with a serine OH nearly eliminated the
binding of mAb 2G7, yet had no effect on LPS binding (see text) or
E. coli LPS-mediated host cell activation (see text). Other
charge reversal mutations in adjacent residues D9, D10, and D12 also
eliminated 2G7 binding, suggesting that this group of charged residues
is located in close proximity on the protein surface. Consistent with
this idea is the observation that serine replacement mutations in
residues D9, D10, and D12 resulted in only a partial reduction in 2G7
binding. Multiple alanine replacement mutations in hydrophilic region 1
have also identified this region as contributing to the epitope of CD14
mAb 3C10 (15). However, in our analysis CD14 mutation
protein E37K, located in hydrophilic region 3, also eliminated 2G7
binding (Table II). The effect of E37K was selective (the epitopes for
MY4 or 6C8 were not affected), consistent with the idea that this
residue is located in close physical proximity to the epitope for 2G7
and did not induce a major conformational change in the protein. This
would indicate that hydrophilic regions 1 and 3 are located in close
enough physical proximity on the CD14 protein surface to influence 2G7
binding.
Consistent with this, the epitope for mAb MY4 also apparently spans hydrophilic regions 1 and 3. CD14 mutation proteins located in hydrophilic region 1 (D9S, D9K, D10K, D12S, and D12K) reduced or eliminated MY4 binding, consistent with an early report demonstrating that a deletion in this region also eliminated binding of this Ab (29). However, a deletion of hydrophilic region 3 (29) and multiple alanine replacement mutations located in region 3 (16) have implicated this region as necessary for MY4 binding. These data are most easily reconciled by placing hydrophilic regions 1 and 3 in close enough physical proximity on the protein surface so that mutations in each region influence MY4 binding.
Epitope mapping of mAb 6C8 indicates that hydrophilic region 1 is also
located in close physical proximity to region 4. A major portion of the
6C8 epitope is located in the hydrophilic region 4 (residues R53 to
D59; see Table II). This region of CD14 has been identified as an
E. coli LPS binding site based upon mutational analysis
(15), mAb binding (15), and protease
protection experiments (14). Residue D59 is probably a
contact residue for Ab 6C8. Similar to residue E11 and Ab 2G7,
replacement of the side chain carboxyl group of D59 with a serine OH
nearly eliminated the binding of mAb 6C8, yet had no effect on LPS
binding (Table II; see text) or E. coli LPS-mediated host
cell activation (see text). A single Ala replacement of residue D59
also eliminates MEM 18 binding, suggesting that 6C8 and MEM 18 share at
least one residue in their epitope (15). However, CD14
mutation proteins D10S, D10K, and R14E, which are located at or near
hydrophilic region 1, also partially or completely reduced the binding
of mAb 6C8. Because D10S had no detrimental effect on LPS binding or
host cell activation, and its only phenotype was a partial reduction in
Ab 2G7 binding, it is likely that this residue is located at or near
the epitope for both Abs. Similarly, although conformational effects
cannot be ruled out, the reduction in 6C8 binding observed for CD14
mutation proteins D10K and R14E are probably due to it being located
close to or in the epitope for Ab 6C8.
What are the structural implications for hydrophilic regions 1, 3, and
4 being located in close enough physical proximity so that residues in
each region influence Ab binding? Ab epitopes on protein surfaces
usually contain six to eight contact residues that bind the
complementarity-determining region of the Ab. The side groups of other
residues on the protein can affect binding to the Ab by steric
hindrance or charge repulsion if they are located in close tertiary
proximity to the contact residues. Because all the anti-CD14 mAbs
examined in this study recognized native, but not denatured protein,
due to the likelihood of discontinuous epitopes, any residue could
theoretically influence Ab binding if it were located in the area
necessary for Ab binding. The surface area on proteins typically
recognized by Abs is 700–900 A2 (30, 31). Therefore, it is safe to conclude that in the native
protein hydrophilic regions 1, 3, and 4 all exist on the same side of
the protein surface.
We therefore propose that CD14 presents to its various microbial ligands an array of different potential binding interactions by orientating several of its groups of charged residues to one side of the protein, in close physical proximity to each other. We suggest that numerous charged residues located throughout the amino-terminal region of CD14 may participate in ligand binding, each contributing to the total energy of binding and accounting for nanomolar concentration binding constants for ligands such as E. coli LPS. This proposal is consistent with the mutational analysis studies presented here and previously (13) that revealed numerous serine or alanine replacement mutations that did not display a difference in binding to E. coli LPS. If, indeed, the binding site consists of numerous residues, then a mutation in any one may not reduce the affinity sufficiently to be measured experimentally. In contrast, charge reversal mutations may interrupt binding by charge repulsion, effectively blocking the LPS ligand from docking.
The selective binding data from CD14 mutation proteins in group 2 indicate that different LPS ligands interact differently with the CD14 LPS binding site. It is tempting to speculate that different LPS binding characteristics of P. gingivalis, H. pylori, and E. coli to CD14 may be related to the different structures and cell-stimulating properties of these LPS species. In addition to differences in core sugar composition, P. gingivalis lipid A differs from E. coli lipid A by the absence of an ester-linked phosphate at the 4' position and fatty acids at the 3 and 3' positions and the presence of fatty acids possessing 16–17 carbon atoms (24). The H. pylori lipid A structure has not been specifically discerned, but compositional analysis has shown that it contains long 3-hydroxy fatty acids (16–18 carbon atoms) and has an unusual phosphorylation pattern compared with that of E. coli lipid A (23). In addition, both P. gingivalis and H. pylori LPS are unusual, in that, unlike E. coli LPS, they are poor stimulators of cytokines from monocytes (32, 33, 34, 35) and do not stimulate E-selectin expression from human endothelial cells (32). However, less potent monocyte activation may be due to poor LBP binding (20), and currently there is no evidence that CD14 LPS binding patterns influence downstream signaling events. Although the possibility that different LPS binding patterns influence downstream signaling events cannot be ruled out, it is more likely that the differences in LPS binding observed here were due to lower binding affinities of these LPS for CD14 compared with E. coli (20). It is possible that the structure of these LPS species does not permit as many interactions with charged residues on CD14, making it easier to detect decreased binding with select CD14 mutation proteins in our assays.
This hypothesis is consistent with the data generated from several different laboratories that have investigated the LPS ligand properties of this molecule. It proposes that LPS ligands bind this charge "catch" region in a nonspecific fashion, i.e., LPS ligands are capable of interacting with a variety of different charge residues. This tactic is similar to that previously reported for the macrophage scavenger protein (19), where the collagenous domain has been proposed to "provide a selectively sticky surface that functions as a kind of molecular flypaper for the high affinity binding of specific polyanions." The hypothesis also provides an explanation for the apparent lack of ligand-specific recognition (12). The exact mechanism by which CD14 binds multiple non-self ligands must await the precise structural determination afforded by protein crystallization. Nevertheless, this hypothesis is consistent with the idea (13) that no one pattern recognition receptor is capable of non-self recognition. Rather, the innate host defense system employs multiple proteins with different binding affinities, transfer properties, and locations to arrive at a consensus about non-self and mount an appropriate response.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LBP, LPS binding protein; sCD14, soluble CD14.
Received for publication August 17, 1999. Accepted for publication January 5, 2000.
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S.-N. Lin, K. Ayada, Y. Zhao, K. Yokota, R. Takenaka, H. Okada, R. Kan, S. Hayashi, M. Mizuno, Y. Hirai, et al. Helicobacter pylori heat-shock protein 60 induces production of the pro-inflammatory cytokine IL8 in monocytic cells J. Med. Microbiol., March 1, 2005; 54(3): 225 - 233. [Abstract] [Full Text] [PDF]
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S. B. Mizel, A. P. West, and R. R. Hantgan Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin J. Biol. Chem., June 20, 2003; 278(26): 23624 - 23629. [Abstract] [Full Text] [PDF]
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M. Muroi, T. Ohnishi, and K.-i. Tanamoto Regions of the Mouse CD14 Molecule Required for Toll-like Receptor 2- and 4-mediated Activation of NF-kappa B J. Biol. Chem., October 25, 2002; 277(44): 42372 - 42379. [Abstract] [Full Text] [PDF]
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 V. J. McCracken, T. Chun, M. E. Baldeon, S. Ahrne, G. Molin, R. I. Mackie, and H. R. Gaskins TNF-{alpha} Sensitizes HT-29 Colonic Epithelial Cells to Intestinal Lactobacilli Experimental Biology and Medicine, September 1, 2002; 227(8): 665 - 670. [Abstract] [Full Text] [PDF]
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K. Watanabe, O. Yilmaz, S. F. Nakhjiri, C. M. Belton, and R. J. Lamont Association of Mitogen-Activated Protein Kinase Pathways with Gingival Epithelial Cell Responses to Porphyromonas gingivalis Infection Infect. Immun., November 1, 2001; 69(11): 6731 - 6737. [Abstract] [Full Text] [PDF]
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M. Hirschfeld, J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody, D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel Signaling by Toll-Like Receptor 2 and 4 Agonists Results in Differential Gene Expression in Murine Macrophages Infect. Immun., March 1, 2001; 69(3): 1477 - 1482. [Abstract] [Full Text] [PDF]
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R. Dziarski, S. Viriyakosol, T. N. Kirkland, and D. Gupta Soluble CD14 Enhances Membrane CD14-Mediated Responses to Peptidoglycan: Structural Requirements Differ from Those for Responses to Lipopolysaccharide Infect. Immun., September 1, 2000; 68(9): 5254 - 5260. [Abstract] [Full Text] [PDF]
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J. da Silva Correia and R. J. Ulevitch MD-2 and TLR4 N-Linked Glycosylations Are Important for a Functional Lipopolysaccharide Receptor J. Biol. Chem., January 11, 2002; 277(3): 1845 - 1854. [Abstract] [Full Text] [PDF]
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), D12K (
), D59K (X), or CD40:Rg (
). After 1
h of incubation at 37 C, LPS bound to sCD14:Rg was detected with mouse
anti-LPS mAbs (see Materials and Methods). The
experiments were performed on three separate occasions, and the
averages from duplicate wells from a representative experiment are
shown.
