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Originally published In Press as doi:10.1074/jbc.M605031200 on January 16, 2007

J. Biol. Chem., Vol. 282, Issue 11, 7877-7884, March 16, 2007
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Endotoxin-binding Proteins Modulate the Susceptibility of Bacterial Endotoxin to Deacylation by Acyloxyacyl Hydrolase*

Theresa L. Gioannini{ddagger}§1, Athmane Teghanemt{ddagger}, DeSheng Zhang{ddagger}, Polonca Prohinar{ddagger}, Erika N. Levis{ddagger}, Robert S. Munford||, and Jerrold P. Weiss{ddagger}**

From the Inflammation Program, Departments of {ddagger}Internal Medicine, §Biochemistry, and **Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, Veterans Affairs Medical Center, Iowa City, Iowa 52246, and ||Department of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, May 25, 2006 , and in revised form, January 8, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444–16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579–36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg) ± LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14 ≥endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1) >>Eagg, Eagg:LBP (E/LBP ~1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue invasion by even minute quantities of many Gram-negative bacteria (GNB)2 initiates rapid mobilization of the innate immune responses of the host. In these circumstances, both GNB recognition and many responses depend upon activation of the exquisitely sensitive Toll-like receptor 4 (TLR4) by endotoxins, structurally unique and abundant glycolipids that occupy much of the outer leaflet of the GNB outer membrane (3). Maximal TLR4-dependent host responses to endotoxin are orchestrated through a sequential set of interactions of endotoxin with lipopolysaccharide-binding protein (LBP), membrane or soluble CD14, and soluble or TLR4-associated MD-2 (3–5). Although timely mobilization of host responses is essential, equally important is the regulation of the duration and intensity of host responses to endotoxin to prevent over-exuberant and sustained responses that can result in severe pathological consequences (6).

One mechanism of dampening host responses to endotoxin is for the host to modify endotoxin itself, converting it from a potent TLR4 agonist to a much weaker agonist with antagonistic properties. To date, the best described host endotoxin-detoxifying enzyme is acyloxyacyl hydrolase (AOAH) (6). AOAH reduces endotoxin activity by catalyzing the release of secondary fatty acyl chains that are attached to primary 3-hydroxy fatty acyl chains within the bioactive lipid A region (7, 8). AOAH thus converts hexaacylated endotoxin species that are potent TLR4 agonists to pentaacylated or tetraacylated forms that have reduced or no agonist properties (9–13) and, at least in vitro, possess the ability to inhibit TLR4 activation by intact, hexaacylated species (10, 11, 13, 14). Recent studies indicate that hexaacylated and partially deacylated or underacylated endotoxin react similarly with LBP, CD14, and MD-2 to form monomeric complexes with MD-2 (13). However, only hexaacylated endotoxin-human MD-2 is a potent TLR4 agonist (13, 14).

AOAH-dependent deacylation of endotoxin has been demonstrated in vivo (1, 15, 16) and in complex in vitro settings that roughly simulate the extracellular and intracellular conditions of inflammatory fluids (1, 2, 15, 17–19). In contrast, either as purified enzyme or as part of a cell lysate, AOAH is virtually inactive against purified or membrane-bound endotoxin in buffered salt solutions in the absence of detergent (20, 21). This suggests that AOAH alone is unable to act efficiently on endotoxin when the fatty acyl chains of the amphipathic endotoxin are buried within pure supramolecular aggregates or the bacterial outer membrane. In contrast, the ability of AOAH to function physiologically suggests that other endotoxin binding molecules may act as "physiological detergents" to facilitate access of AOAH to the acyloxyacyl bonds that link the non-hydroxylated fatty acyl chains to lipid A.

The delivery of endotoxin to MD-2, as is needed for TLR4 activation, is markedly enhanced by LBP- and CD14-dependent extraction and transfer of endotoxin monomers from purified endotoxin aggregates (Eagg) (4) or the bacterial outer membrane (22). These steps are also albumin-dependent (23), suggesting that Eagg (or membrane-bound endotoxin)-LBP and monomeric endotoxin-CD14 intermediates present endotoxin in a manner that partially exposes the fatty acyl chains and so require albumin to maintain endotoxin solubility in an aqueous environment. By making use of metabolically radiolabeled endotoxin from Neisseria meningitidis serotype B (24) and from Escherichia coli, we now show that AOAH can act on endotoxin-LBP aggregates and, to an even greater extent, on monomeric endotoxin-sCD14 complexes, but much less on monomeric endotoxin-MD-2. These findings strongly support a model in which LBP- and CD14-dependent extraction and transfer of endotoxin expose fatty acyl chains that then become less accessible when endotoxin binds MD-2. The ability of AOAH to act on these protein-endotoxin intermediates provides a mechanism by which partial deacylation of endotoxin can take place before transfer to MD-2 and thus down-regulate TLR4 activation by endotoxin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
LBP, BPI, and sCD14 were provided by Xoma (US) LLC (Berkeley, CA). Recombinant soluble human MD-2 containing a hexapolyhistidine tag on the carboxyl-terminal end was prepared using infection of High Five insect cells with baculovirus containing the cDNA for human MD-2 as described (25). Recombinant human AOAH was a generous gift from Zymo-Genetics (Seattle, WA). Chromatography matrices were purchased from GE Healthcare. Human serum albumin (HSA) was obtained as an endotoxin-free, 25% stock solution (Baxter Healthcare Corp., Glendale, CA). A soluble truncated CD14 (amino acids 1–156) containing a hexapolyhistidine tag at the carboxyl terminus was generated by infection of High Five insect cells with baculovirus containing the cDNA for this protein derived from recombination with the vector pBAC11 (Novagen) using the procedure previously described for soluble MD-2 (25). Insect supernatant containing sCD14-(1–156) was dialyzed against 20 mM phosphate, pH 7.4, 0.5 M NaCl before adsorption to Ni Fast Flow-Sepharose resin (GE Healthcare). The protein was purified on the AKTA Explorer 100 FPLC using an imidazole gradient for elution of adsorbed protein. HisLink resin (Promega) was used analytically to capture complexes by the method previously described (25). Note no imidazole was used in the binding buffer or rinses but buffer did contain 0.1% bovine serum albumin.


Figure 1
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  		FIGURE 1.
Physical presentation of LOS affects its susceptibility to deacylation by AOAH. A, [3H]LOSagg and LOS-sCD14 protein complexes (each corresponds to 1–2 nM) were treated for 2 h at 37 °C with AOAH (750 pM; see "Experimental Procedures") in HBSS+, pH 7.4, 10 mM HEPES, 0.1% HSA. Partial deacylation of [3H]LOS was measured as described under "Experimental Procedures." B, recombinant LBP or BPI at varying concentrations (0.1 pM-10 nM) was incubated with LOSagg (1–2 nM) for 20 min at 37 °C to form LOSagg-protein complexes and then treated with AOAH (750 pM) for 2 h at 37 °C, followed by assay of free 3H-fatty acid released. Data are plotted as the molar ratio of either LBP or BPI to LOS shown as % deacylation with release of 33.3% of [3H]LOS cpm as free fatty acids corresponding to 100% deacylation (see "Experimental Procedures"). C, [3H]LOSagg (1 nM) was incubated with increasing concentrations of LBP (0.2 pM-1 nM) and sCD14 (5 nM) in 1 ml of HBSS+, pH 7.4, 10 mM HEPES, 0.1% HSA for 30 min at 37 °C and analyzed by gel filtration chromatography on Sephacryl S200 as described under "Experimental Procedures" for formation of LOS-sCD14, Mr ~ 60,000. Results are reported as % of LOS converted to LOS-sCD14 complex. In each experiment, recovery of applied radioactivity was ≥90% as judged by liquid scintillation spectroscopy. Data shown represent either the mean ± S.E. of three or more independent determinations (A and B) or mean of two determinations (C).

 
Preparation of Metabolically Labeled Endotoxin and Endotoxin-Protein Complexes—[3H]LOS (specific activity 25,000 cpm/pmol) was isolated from an acetate auxotroph of N. meningitidis serogroup B after metabolic labeling as previously described (24). LOSagg (apparent Mr > 20 million) used as starting material to generate complexes was prepared using Sephacryl HR S500 or by ultracentrifugation (24). Aggregates containing various concentrations of LBP (LOSagg-LBP) or BPI (LOS-BPIagg) to be used in AOAH assays were prepared in situ (0.1 ml volume) by incubation of 1 nM [3H]LOSagg in Hanks' buffered salt solution with Ca2+ and Mg2+ (HBSS+), pH 7.4, 10 mM HEPES, 0.1% HSA for 20 min with the indicated concentration of either LBP or BPI (range from 0.1 to 10 nM) before addition of AOAH.

[3H]LOS-sCD14, [3H]LOS-sCD14-(1–156), and [3H]LOS-MD-2 complexes were isolated by gel filtration chromatography using columns of Sephacryl HR S200 (1.6 x 30 cm or 1.6 x 70 cm) equilibrated in HBSS+, pH 7.4, 10 mM HEPES, ± 0.1% HSA. Isolated [3H]LOS-sCD14 (Mr ~ 60,000) or [3H]LOS-sCD14-(1–156)-His6 (Mr ~ 25,000) complexes were generated by incubation at 37 °C for 30 min of LBP with LOSagg (usual molar ratio 1:100) and either equimolar sCD14 (23, 24) or purified sCD14-(1–156)-His6 before isolation by gel filtration chromatography on Sephacryl HR S200 in HBSS+, pH 7.4, 10 mM HEPES, 0.03% HSA. In experiments where the effects of LBP concentration on formation of [3H]LOS-sCD14 were evaluated, the concentration of LBP ranged from 0.2 pM to 1 nM with a concentration of 1 nM [3H]LOS. Evaluation of the formation of monomeric [3H]LOS-sCD14 was monitored by gel filtration chromatography on Sephacryl HR S200 in PBS, pH 7.4, 0.03% HSA. [3H]LOS-MD-2 was prepared by treatment for 30 min at 37 °C of [3H]LOS-sCD14 with MD-2-containing insect culture medium followed by isolation of the monomeric [3H]LOS-MD-2 by Sephacryl HR S200 chromatography.


Figure 2
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  		FIGURE 2.
Quantitative analysis of AOAH activity toward LOS-sCD14 and LOS aggregates. Increasing amounts of [3H]LOSagg or [3H]LOS-sCD14 in HBSS+, pH 7.4, 10 mM HEPES, 0.1% HSA were incubated with AOAH (750 pM) in a volume of 0.1 ml for 2 h at 37 °C before ethanol precipitation of LOS and determination of released (ethanol-soluble) 3H-free fatty acids. Data shown correspond to mean ± S.E. and are representative of three similar experiments, each in duplicate. Inset represents Lineweaver-Burk analysis of data from the representative curve. Analysis was done using Graphpad Prism 4 programs.

 
For determination of the ability of AOAH-treated [14C]LOS-sCD14 to transfer partially deacylated [14C]LOS to MD-2, [14C]LOS-sCD14 (40 nM) was treated with AOAH (concentration adjusted based on method as described below) for 4 h at 37 °C. An aliquot (10 µl) was removed to evaluate the extent of deacylation by measuring ethanol-soluble radioactivity (i.e. 14C-free fatty acids) described below. The remainder of the sample was incubated with insect culture medium containing MD-2-His6 for 30 min at 37 °C. The reaction mixture was resolved on Sephacryl HR S200 in PBS, pH 7.4, and the fractions corresponding to [14C]LOS-MD-2 were subjected to 14C-fatty acid analysis. [14C]lipopolysaccharide (LPS) (specific activity ~6,000cpm/pmol) was isolated from an acetate auxotroph generated in E. coli BW25113 (BW25113 aceE). This strain was created by deletion-insertion mutagenesis according to the method of Datsenko and Wanner (26) resulting in insertion of a chloramphenicol acetyltransferase (cat) gene cassette within the aceE gene. Disruption of the aceE gene and insertion of the chloramphenicol acetyltransferase cassette was confirmed by PCR analysis and growth was shown to be acetate-dependent. Analysis of the 14C-fatty acids released by acid and alkaline hydrolysis of isolated LPS was performed as described below for LOS to determine the specific activity and composition of 14C-fatty acids in the isolated LPS.3 [14C]LPSagg, [14C]LPS-sCD14, and [14C]LPS-MD-2 complexes were prepared as described above for [3H]LOS.

In gel filtration chromatography, fractions (1 ml) were collected (flow rate 0.5 ml/min) at room temperature using AKTA PurifierTM FPLC. Aliquots of the collected fractions were analyzed by liquid scintillation spectroscopy using a Beckman LS liquid scintillation counter to detect radioactive LOS. Recoveries of LOS and LPS were >70%. All solutions used were pyrogen-free and sterile-filtered. After chromatography, selected peak fractions to be used in bioassays were pooled and passed through sterile syringe filters (0.22 or 0.45 µm) with greater than 90% recovery of radiolabeled material in the sterile filtrate. Fractions were stored under sterile conditions at 4 °C until needed. Sephacryl S200 columns were calibrated with Bio-Rad gel filtration standards that included thyroglobulin (Vo), {gamma}-globulin, human serum albumin, ovalbumin, myoglobin, and vitamin B12 (Vi).

AOAH Deacylation Assay—AOAH was stored at –80 °C, diluted in storage buffer (20 mM sodium acetate, pH 6, 0.9% NaCl, 25% glycerol, 0.05% Triton X-100) (27) immediately prior to usage and added to samples as indicated. The volume of AOAH in storage buffer added to samples was <1% of the total incubation volume. In addition to samples prepared in HBSS+, pH 7.4, 10 mM HEPES, 0.1% HSA, LOSagg was incubated with AOAH in 20 mM sodium acetate, pH 6, 150 mM NaCl, 5 mM CaCl2, 0.1% bovine serum albumin, 0.1% Triton X-100 (27) as a positive control for AOAH activity. AOAH activity (1 microunit hydrolyzes 1 pmol fatty acid/min under the above detergent, acidic pH conditions) was assayed by determining release of radioactively labeled free fatty acids from metabolically labeled LOS or LPS. Duplicate samples (0.1 ml) of ~1 nM [3H]LOS or 2 nM [14C]LPS were incubated with AOAH (750 pM, 15 microunits) for [3H]LOS or [14C]LPS for 2 h at 37 °C unless otherwise indicated. This amount of AOAH used/mol LOS (LPS) is similar to the ratios of enzyme and substrate previously used under detergent/acidic conditions (27). The quantity of fatty acid released was evaluated using the ethanol precipitation method (27) wherein undigested and partially deacylated endotoxin are precipitated and released free fatty acids remain in the ethanol supernatant. Briefly, after incubation ± AOAH, samples (100 µl) were treated with 0.2 ml of ice-cold ethanol and kept at –20 °C overnight. Samples were spun for 10 min at 14,000 x g. Supernatants were removed; the pellets were washed with an additional 0.2 ml of ice-cold ethanol, incubated for 1 h at 0 °C, and spun for 10 min at 14,000 x g. The supernatant and rinses were combined for analysis of released radiolabeled free fatty acids by liquid scintillation spectroscopy. Pellets were treated with 0.2 ml of 2% SDS for 30 min at 37 °C and then also evaluated by liquid scintillation spectroscopy. Overall recovery of radioactivity was ≥90%. The results of reaction with AOAH are reported as % deacylation. Because only two of six moles of fatty acid of LOS/LPS attached to lipid A can be released by AOAH, 33% fatty acid release was defined as 100% deacylation.


Figure 3
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  		FIGURE 3.
LOS in a complex with either full-length or a truncated form of CD14 is sensitive to deacylation by AOAH and remains associated with CD14 after partial deacylation. A, [3H]LOS-sCD14 (4 nM) or LOS-sCD14-(1–156)-His6 (3 nM)(Mr ~ 25,000) was incubated with AOAH (750 pM) at 37 °C for 2 or 4 h, and release of free fatty acids was analyzed as described under "Experimental Procedures." B, [3H]LOS-sCD14-(1–156) (4 nM) was incubated ± AOAH (750 pM) for 4 h at 37 °C. An aliquot (100 ul) was analyzed for the degree of deacylation by ethanol precipitation, and the remainder of the sample was applied to Sephacryl S200 in PBS, 0.03% HSA, pH 7.4. Fractions were analyzed by liquid scintillation spectroscopy. Data shown are from one analysis, representative of three experiments. C, isolated AOAH-treated [3H]LOS-sCD14-(1–156)-His6 (Mr ~ 25,000), as well as a comparable sample of untreated [3H]LOS-sCD14-(1–156)-His6, were incubated with HisLink resin (100 µl) equilibrated in PBS, pH 7.4, 0.03% HSA, for 1 h at 25 °C with gentle mixing. The flow-through was collected, beads were washed with PBS, pH 7.4, 0.03% HSA, and the adsorbed material eluted with 2% SDS. [3H]LOS in flow-through, rinses, and bead eluate were analyzed by liquid scintillation spectroscopy. Total recovery of [3H]LOS was ≥90%. Data shown represent either the mean ± S.E. of four separate determinations (A) or correspond to one experiment, representative of three or more experiments (B and C) with duplicate samples.

 
Fatty Acid Analysis—The 14C fatty acid content of various 14C-endotoxin and 14C-endotoxin-protein preparations was analyzed as previously described (22, 24). In brief, these complexes were subjected to acid hydrolysis (4 N HCl, 90 °C), neutralization, and base hydrolysis (4 N NaOH), followed by Bligh-Dyer extraction and recovery of the released 14C-labeled fatty acids in the chloroform phase. The 14C-fatty acids were resolved by reversephase TLC (0.2 mm HPTLC, RP-18; Merck) using acetonitrile/acetic acid (1/1) as the solvent system. 14C-fatty acids were identified by comparison to migration of 14C-fatty acid standards. Analysis was done using the Typhoon imaging system (GE Healthcare).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LBP and sCD14 Increase LOS Susceptibility to AOAH—To determine whether LBP- and/or CD14-dependent alterations of endotoxin presentation render endotoxin more sensitive to AOAH, we examined the ability of AOAH to deacylate purified meningococcal LOS when LOS was presented either alone, as purified LOSagg, after treatment with LBP to produce LOSagg-LBP aggregates, or after sequential treatment with LBP and sCD14 to yield monomeric LOS-sCD14 complexes. All assays were performed in HBSS+, 10 mM HEPES, 0.1% HSA, pH 7.4. The susceptibility of LOS to AOAH was markedly increased by pretreatment of LOSagg with LBP + sCD14 to convert LOSagg to monomeric LOS-sCD14 complex (Fig. 1A) (23). Incubation of LOSagg with LBP alone also markedly increased the susceptibility of LOS to AOAH at specific LBP:LOS ratios (Fig. 1B). The dose-dependence of LBP alone on AOAH susceptibility of LOSagg showed a complex bell-shaped curve (Fig. 1B) with maximum AOAH activity at a molar ratio of ~1 mol LBP/100 mol LOS. At the concentration of LBP most favorable for AOAH activity, there was no detectable change in the gel sieving profile of the LOSagg (i.e. no detectable disaggregation of LOSagg) (28). The bell-shaped LBP dose-dependence of AOAH activity on LOSagg closely paralleled the bell-shaped LBP dose-dependence for extraction and delivery of LOS monomers to sCD14 (Fig. 1C). This suggests that LBP-catalyzed extraction and delivery of endotoxin monomers to sCD14 produce alterations in the orientation of endotoxin within LOSagg that favor access of AOAH to endotoxin, independent of CD14. In contrast to the effects of LBP, the closely related endotoxin-binding protein bactericidal permeability increasing protein (BPI) did not promote formation of LOS-sCD14 (28) or significantly increase LOSagg susceptibility to AOAH (Fig. 1B).


Figure 4
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  		FIGURE 4.
Partially deacylated monomeric [14C]LOS-MD-2 complex containing LOS is generated from [14C]LOS-sCD14 treated with AOAH. A, [14C]LOS-sCD14 (40 nM) was incubated with AOAH (3.75 nM) for 4 h at 37 °C in 0.5 ml of HBSS+, 10 mM HEPES, 0.1% HSA. Ethanol precipitation of an aliquot (10 ul) indicated release of ~20% of [14C]LOS cpm as free fatty acids (i.e. partial deacylation of 60% of [14C]LOS-sCD14). AOAH-treated [14C]LOS-sCD14 was incubated with High Five insect medium containing MD-2-His6 for 30 min at 37 °C, and the reaction mixture was resolved on Sephacryl HR S200 (1.6 x 70 cm) in PBS, pH 7.4. Elution of monomeric LOS-sCD14, LOS-MD-2, and aggregates of LOS-sCD14 (Vo) formed during the incubation at 37 °C is indicated. B, 14C-fatty acid content of [14C]LOS-MD-2 recovered from Sephacryl S200 chromatography (see encircled fractions in panel A) after incubation of untreated or AOAH-treated LOS-sCD14 with MD-2 was determined as described under "Experimental Procedures." The relative amounts of individual fatty acid species were determined by image analysis using the Typhoon imager. The figure shown represents the results of one of two similar experiments.

 
Under optimal conditions, the susceptibility of LOSagg-LBP (100:1 mol/mol) and LOS-sCD14 to AOAH appeared to be similar (Fig. 1, A and B). We focused further biochemical characterization on the substrate properties of LOS-sCD14 because of the greater molecular homogeneity of LOS-sCD14 in comparison to LOSagg-LBP. Fig. 2 compares the rate of fatty acid release versus substrate concentration for two substrates, LOS-sCD14 and LOSagg. The data demonstrate a >10-fold increase in AOAH sensitivity conferred by extraction and delivery of endotoxin monomers from LOSagg to LOS-sCD14. Lineweaver-Burk analysis of the data for LOS-sCD14 indicated that the Km is ~5 nM and the Vmax is 9.1 ± 0.05 fmol/min (n = 3).

Reaction of LOS-sCD14 with AOAH Yields Partially Deacylated LOSAOAH-sCD14 Complex That Can React with MD-2 to Generate LOSAOAH-MD-2—The ability of LBP and CD14 to promote AOAH-dependent deacylation of LOS suggested a mechanism by which non-covalent host protein-mediated modifications in lipid A presentation might facilitate not only TLR4 activation but also AOAH-mediated lipid A deacylation that can reduce TLR4 activation (9–14). Underacylated and partially deacylated endotoxin can be transferred from CD14 to MD-2 (13, 14, 29) and the underacylated AOAH-treated endotoxin-MD-2 complex interacts with TLR4 without efficiently inducing TLR4 activation (13). Thus, provided the partially deacylated LOS remains bound to CD14 during and after AOAH treatment, deacylation of monomeric endotoxin-sCD14 complex (e.g. LOS-sCD14) could provide substrate for generation of AOAH-treated endotoxin-MD-2 complex (e.g. LOSAOAH-MD-2) that can then act as a TLR4 antagonist.

Whether or not deacylated LOS remained associated with sCD14 could not be tested readily by comparing the gel sieving properties of the LOS-sCD14 complex before and after treatment with AOAH because of the closely similar Mr of sCD14 and AOAH. Therefore, we made use of a truncated form of recombinant CD14 (amino acid residues 1–156) containing a carboxyl-terminal hexapolyhistidine tag (sCD14-(1–156)-His6). This recombinant sCD14-(1–156)-His6 fully retains the reactivity of full-length sCD14 (356 residues) toward LOSagg-LBP and MD-2 (data not shown). LOS-sCD14-(1–156)-His6 was also a good substrate for AOAH (Fig. 3A). After AOAH treatment, the majority of the [3H]LOS cpm remained associated with sCD14-(1–156)-His6 as judged by gel sieving analysis (Mr ~ 25,000) (Fig. 3B) and co-capture of [3H]LOSAOAH contained in the Mr ~ 25,000 peak by metal chelation chromatography (Fig. 3C). The earlier eluting peaks containing [3H]LOS-derived cpm may represent dimers and larger aggregates of [3H]LOSAOAH-CD14-(1–156)-His64 as well as released 3H-free fatty acids complexed with albumin.

To verify that treatment of LOS-sCD14 with AOAH yielded partially deacylated LOS-sCD14 complex capable of transferring the partially deacylated LOS to MD-2, [14C]LOS-sCD14 (full-length sCD14) was deacylated with AOAH, subsequently treated with MD-2-His6, and the product(s) separated using Sephacryl S200 (Fig. 4A). Fatty acid analysis of the peak fractions of [14C]LOS corresponding to [14C]LOS-MD-2 by migration on Sephacryl S200 indicated that the recovered LOS-MD-2 contained partially deacylated LOS (Fig. 4B). Experimental conditions were used that yielded ~60% release of the non-hydroxylated fatty acid (12:0) from LOS-sCD14 (data not shown). Because cleavage by AOAH of the two non-hydroxylated fatty acids present in each molecule of endotoxin normally follows in rapid succession (2, 19, 27), the major product under our experimental conditions is likely tetraacylated LOS-sCD14 with ~40% of the hexaacylated LOS-sCD14 exposed to AOAH remaining undigested. The close match in 14C-fatty acid composition of the AOAH-treated [14C]LOSAOAH-sCD14 and the recovered [14C]LOSAOAH-MD-2 suggests, therefore, essentially equal efficiency of transfer of undigested and partially deacylated LOS from sCD14 to MD-2, as was previously reported (13).


Figure 5
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  		FIGURE 5.
Endotoxin-sCD14 is more sensitive to deacylation by AOAH than endotoxin-MD-2 or endotoxin aggregates. A, [3H]LOSagg, [3H]LOS-MD-2, and [3H]LOS-sCD14 (1 n M) were treated with increasing concentrations of AOAH in 0.1 ml for 2 h at 37 °C. After incubation, samples were ethanol precipitated and released free fatty acids were measured as described under "Experimental Procedures." Data shown represent the mean ± S.E. of at least three determinations. B, [14C]LPSagg, [14C]LPSagg-LBP at the indicated LBP-LPS molar ratios, [14C]LPS-sCD14, and [14C]LPS-MD-2 (2–3 nM) were treated with AOAH (750 pM) in 0.1 ml of HBSS+, 10 mM HEPES, 0.1% HSA for 2 h at 37 °C. After incubation, samples were ethanol precipitated and released free fatty acids were measured as described under "Experimental Procedures." Data shown represent the mean ± S.E. of two or more experiments, each with duplicate samples.

 
Monomeric Endotoxin-MD-2 Complex Is Relatively Resistant to AOAH—In contrast to LOS-sCD14, monomeric LOS-MD-2 complexes do not depend upon albumin to maintain aqueous solubility and induce TLR4-dependent cell activation. Therefore, we have speculated that the fatty acids of lipid A are more fully buried within a deeper hydrophobic cavity within MD-2 and so might be a poorer substrate for AOAH. To test this hypothesis, we compared the effect of increasing concentrations of AOAH on LOS-sCD14 and LOS-MD-2 and, for comparison, unmodified LOSagg. As predicted, LOS-MD-2, in comparison to LOS-sCD14, showed much reduced sensitivity to added AOAH (Fig. 5A). As illustrated in Fig. 5B, a closely similar pattern of endotoxin sensitivity to AOAH comparing purified endotoxin aggregates (LPSagg) ± LBP and monomeric LPS-sCD14 or MD-2 was seen with a rough form of E. coli [14C]LPS isolated from E. coli strain BW25113 aceE. Monomeric LPS-sCD14 complex and LPSagg-LBP at substoichiometric ratios of LBP-LPS, which most readily resulted in the formation of LPS-sCD14, were deacylated most effectively by AOAH in HBSS+, 10 mM HEPES, 0.1% HSA, pH 7.4. As seen with LOS, LPS in the form of LPSagg and LPS-MD-2 was much more resistant to deacylation by AOAH (Fig. 5B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
LBP and CD14 have important and complex roles in host interactions with endotoxin. The most potent activation of mammalian cells by many endotoxins requires sequential protein-endotoxin and protein-protein interactions involving LBP, C14, MD-2, and TLR4 (3–5). The combined action of LBP and CD14 results in extraction of endotoxin monomers from purified endotoxin aggregates (30–32) and the GNB outer membrane to yield a monomeric endotoxin-CD14 complex that is the preferred substrate of MD-2/TLR4 (4, 25, 33, 34). Maximal generation of endotoxin-CD14 requires a very low molar ratio of LBP to endotoxin and CD14 (Fig. 1C) (30–32). Higher molar ratios of LBP to endotoxin and CD14 reduce MD-2/TLR4-dependent cell activation by endotoxin (31, 32, 35, 36) and instead promote cellular and extracellular (e.g. plasma lipoprotein-dependent) clearance of endotoxin (32, 35, 37–39).

The studies described here demonstrate a new role for LBP and CD14 in negative regulation of the pro-inflammatory action of endotoxin: they can participate as cofactors in the catalytic detoxification of endotoxin by AOAH. Our findings clearly show that both LBP alone and LBP and sCD14 in concert (i.e. by yielding endotoxin-sCD14) substantially increase the sensitivity of both meningococcal LOS and E. coli LPS to AOAH (Figs. 1 and 5). The stimulatory effects of LBP ± sCD14 on AOAH activity require substoichiometric concentrations of LBP (Figs. 1B and 5B) that correspond closely to the concentrations required for LBP-dependent extraction and delivery of endotoxin monomers from endotoxin aggregates to sCD14 (Fig. 1C). The increased sensitivity to AOAH of endotoxin aggregates treated with very low concentrations of LBP suggests that, under these conditions, LBP induces a rearrangement of endotoxin within the aggregates that increases exposure of the lipid A region of endotoxin, facilitating AOAH action as well as extraction and transfer of endotoxin monomers to CD14. The inhibitory effect of higher concentrations of LBP on AOAH action and formation of endotoxin-sCD14 monomers (Fig. 1, B and C) may reflect increased competition by added LBP on the interaction of endotoxin with CD14 or AOAH. The inability of BPI to significantly increase endotoxin sensitivity to AOAH (Fig. 1B) mirrors its inability to promote extraction and transfer of endotoxin to CD14 (28, 40). This may reflect the higher affinity of BPI for endotoxin and further illustrates differences in the interactions of LBP and BPI with endotoxin-rich interfaces (28, 41).

The sensitivity of monomeric endotoxin-sCD14 complex to AOAH is consistent with structural data derived from x-ray crystallography of mouse CD14 (42). Structural and functional data suggest that the binding site for endotoxin in CD14 includes a wide, flexible hydrophobic pocket near the amino terminus (42). The characteristics of this pocket predict that the fatty acyl chains of bound endotoxin will be partially exposed, explaining the need for albumin to maintain the stability of endotoxin-sCD14 in aqueous solution and rendering the acyloxyacyl linkages in lipid A accessible to AOAH. The relative resistance of LOS-MD-2 and LPS-MD-2 to AOAH (Fig. 5, A and B), by contrast, suggests a more complete sequestration of the fatty acyl chains within MD-2. This is consistent with models of MD-2 structure that suggest a deep hydrophobic cavity (43, 44) and with the solubility and stability of endotoxin-MD-2 in aqueous solution even in the absence of albumin (13).

AOAH action on endotoxin-CD14 yields a partially deacylated endotoxin-CD14 complex (Fig. 3, B–D). AOAH-treated endotoxin can be readily transferred from CD14 to MD-2 (see Fig. 4) (13), producing a complex of underacylated endotoxin with MD-2 that can occupy TLR4 without efficiently inducing receptor activation (13, 34). Thus, the underacylated endotoxin-MD-2 complex inhibits responsiveness of TLR4 by competing with the fully hexaacylated endotoxin-MD-2 complex (13). Soon after bacterial invasion, however, levels of extracellular AOAH are likely to be limiting. Moreover, comparison of the reactivity of LOS-sCD14 with AOAH (Km ~ 5 nM; Fig. 2) versus MD-2 (apparent "Km" for transfer ~100 pM) (34) strongly suggests that at low (pM) concentrations of endotoxin-CD14, AOAH is unlikely to blunt MD-2/TLR4-dependent cell activation by endotoxin. This difference in reactivity between AOAH and MD-2 with endotoxin-CD14 may be necessary to permit induction of robust host innate and adaptive immune response to small numbers of invading GNB and pM endotoxin (15). However, under conditions when the levels of endotoxin-sCD14 produced substantially exceed those of MD-2/TLR4 and the concentrations of other potential host reactants with endotoxin-sCD14 (e.g. plasma lipoproteins) are limiting, AOAH activity against endotoxin-CD14 may help constrain potentially protracted endotoxin-driven inflammatory and immune responses.

It should be noted that the experimental conditions we have used roughly model extracellular AOAH action (pH 7.4) against extracellular endotoxin or, perhaps, cell surface-bound monomeric endotoxin-membrane CD14. However, within an inflammatory exudate, intracellular AOAH action within phagocytic cells (macrophages, dendritic cells, neutrophils) may predominate (2, 15, 45, 46). In these cells, most endotoxin that is internalized is taken up in the form of large aggregates or membrane remnants, distinct from the endotoxin-sCD14 complex that is the preferred substrate for MD-2/TLR4 and extracellular AOAH. The identities of the intracellular factors and conditions that promote intracellular AOAH action remain to be determined, as does the biological significance of the partially deacylated endotoxin that is released from phagocytes into their extracellular environment.


   FOOTNOTES
 
* This work was supported by U.S. Public Health Service Grants PO144642 and AI59372 (to J. P. W.) and AI18188 (to R. S. M.) and a Veterans Affairs Merit Review grant (to T. L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Roy J. and Lucille A. Carver College of Medicine, University of Iowa and the Veterans Affairs Medical Center, Inflammation Program, Oakdale Research Campus, 2501 Crosspark Rd., Coralville, IA 52241. E-mail: theresa-gioannini{at}uiowa.edu.

2 The abbreviations used are: GNB, Gram-negative bacteria; AOAH, acyloxyacyl hydrolase; BPI, bactericidal permeability increasing protein; Eagg, endotoxin aggregate; HBSS+, Hanks' buffered salt solution; HSA, human serum albumin; LBP, lipopolysaccharide-binding protein; LOS, lipooligosaccharide; LPS, lipopolysaccharide; sCD14, soluble CD14; TLR4, Toll-like receptor 4; PBS, phosphate-buffered saline. Back

3 P. Prohinar, DeS. Zhang, and J. P. Weiss, unpublished data. Back

4 T. L. Gioannini, data not shown. Back


   ACKNOWLEDGMENTS
 
We thank Xoma Corp. (Berkeley, CA) for recombinant LBP, BPI, and sCD14. We also gratefully acknowledge Nathan Coussens, Dept. of Biochemistry, University of Iowa, for construction of the pBAC11 plasmid containing truncated sCD14.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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