A host lipase detoxifies bacterial lipopolysaccharides in the liver and spleen

Much of the inflammatory response of the body to bloodborne Gram-negative bacteria occurs in the liver and spleen, the major organs that remove these bacteria and their lipopolysaccharide (LPS, endotoxin) from the bloodstream. We show here that LPS undergoes deacylation in the liver and spleen by acyloxyacyl hydrolase (AOAH), an endogenous lipase that selectively removes the secondary fatty acyl chains that are required for LPS recognition by its mammalian signaling receptor, MD-2-TLR4. We further show that Kupffer cells produce AOAH and are required for hepatic LPS deacylation in vivo. AOAH-deficient mice did not deacylate LPS and, whereas their inflammatory responses to low doses of LPS were similar to those of wild type mice for approximately 3 days after LPS challenge, they subsequently developed pronounced hepatosplenomegaly. Providing recombinant AOAH restored LPS deacylating ability to Aoah(-/-) mice and prevented LPS-induced hepatomegaly. AOAH-mediated deacylation is a previously unappreciated mechanism that prevents prolonged inflammatory reactions to Gram-negative bacteria and LPS in the liver and spleen.     

Deacylation of structurally diverse lipopolysaccharides by human acyloxyacyl hydrolase

Acyloxyacyl hydrolase, a leukocyte enzyme previously has been shown to catalyze the hydrolysis of secondary (acyloxyacyl-linked) fatty acyl chains from the nonreducing glucosamine of the lipid A region of rough Salmonella typhimurium lipopolysaccharide (LPS). We describe here the activity of this enzyme toward smooth S. typhimurium LPS and LPS from Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Acyloxyacyl hydrolase released the secondary acyl chains from all of these lipopolysaccharides, regardless of the location of the acyloxyacyl linkage on the diglucosamine backbone or the structure of the acyl chains. The two acyloxyacyl linkages present in each LPS molecule apparently were hydrolyzed separately, so that free fatty acids released from the different sites accumulated at different rates. The purified enzyme also removed greater than 90% of the secondary acyl chains in each LPS, indicating that the enzyme acts not only on intact LPS but also on LPS molecules that have only one secondary acyl chain. The enzyme did not release the glucosamine-linked 3-hydroxyacyl chains. The specificity and versatility of the enzyme for cleaving acyloxyacyl linkages suggest that it may be a useful reagent for studying the structure and bioactivities of lipopolysaccharides with diverse carbohydrate and lipid A structures.     

Acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides, has phospholipase, lysophospholipase, diacylglycerollipase, and acyltransferase activities in vitro.

Human acyloxyacyl hydrolase (AOAH) is a leukocyte enzyme that hydrolyzes acyloxyacyl bonds in the lipid A region of bacterial lipopolysaccharide (LPS), thereby detoxifying the LPS. We report here that the enzyme also acts in vitro on glycerophospholipids, lysophospholipids, and diacylglycerol. While AOAH preferentially removes palmitate or stearate from the sn-1 position of phospholipid and diacylglycerol substrates that have unsaturated acyl chains in the sn-2 position, it is able to cleave both palmitates from sn-1,2-dipalmitoylphosphatidylcholine and sn-1,2-dipalmitoylglycerol. This apparent preference for removing saturated (or shorter) acyl chains from glycerolipids is consistent with its ability to cleave laurate more rapidly than palmitoleate from lipopolysaccharide (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449). AOAH also catalyzes acyl transfer from LPS and phosphatidylethanolamine to acceptor lipids; approximately equal amounts of laurate and myristate are transferred from LPS to monooleoylglyceryl ether, forming acyloleoylglyceryl ether. The demonstration that AOAH has phospholipase, lysophospholipase, diacylglycerol lipase, and acyltransferase activities in vitro suggests that the enzyme may have roles in addition to LPS deacylation (detoxification) in phagocytic cells.     

Expression and characterization of recombinant human acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides

The molecular cloning and eukaryotic cell expression of the complementary DNA for human neutrophil acyloxyacyl hydrolase (AOAH) are described. AOAH is a leukocyte enzyme that selectively removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides (endotoxins), thereby detoxifying the molecules. The two disulfide-linked subunits of the enzyme are encoded by a single mRNA. The amino acid sequence of the protein contains a lipase consensus sequence in the large subunit and a region in the small subunit that is similar to the saposins, cofactors for sphingolipid hydrolases. The recombinant enzyme, like native AOAH, hydrolyzes secondary acyl chains from more than one position on the lipopolysaccharide backbone. Acyloxyacyl hydrolase is a novel two-component lipase that, by deacylating lipopolysaccharides, may modulate host inflammatory responses to Gram-negative bacterial invasion.     

Characterization of a protein fatty acylesterase present in microsomal membranes of diverse origin

A microsomal activity of baby hamster kidney cells which cleaves ester-type bound fatty acids from acyl proteins in vitro has been characterized. This activity is also present in microsomal membranes from pig liver, calf kidney, and human mucous cells. Cell free deacylation is described for the Semliki Forest virus acyl proteins E1 and E2 and the precursor of E2 designated p62. Acyl chain cleavage operates with both exogenous and endogenous viral acyl protein substrates. The in vitro cleavage requires microsomes solubilized by detergents of which various kinds are equally effective (Nonidet P-40, Tween 20, sodium deoxycholate, Triton X-100, or octyl-beta-D-glucoside). If microsomes are boiled for 15 min prior to the incubation, deacylation is abolished completely and no radioactivity is released from the palmitoylated acyl proteins during incubation with either detergents or microsomes alone. No changes in the molecular structure of the deacylated Semliki Forest virus proteins were detected, and the cleavage product was identified as free fatty acid. Deacylation is time- and temperature-dependent and can be enhanced by increasing the concentration of microsomal protein in the incubation mixture. It is completely inhibited under acidic conditions (pH 5) and at low temperature (4 degrees C). Deacylation also occurs in the presence of EDTA and bivalent cations such as Mg2+, Mn2+, and Ca2+ which influence the reaction marginally. On the other hand, fatty acid release is drastically reduced with a mixture of Co2+, Zn2+, and Hg2+ ions. The activity is not identical with protein fatty acyltransferase operating in the reverse direction, since a partially purified preparation of this acyltransferase failed to cleave fatty acids from fatty acylated substrate proteins. Taken together, these data lead us to postulate an enzymatic activity which cleaves fatty acids from ester-type fatty acylated proteins, and we propose to designate this enzyme a protein fatty acylesterase.     

Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS. The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.     

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.     

The transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo

The extraordinary potency and pathological relevance of gram-negative bacterial LPSs have made them very popular experimental agonists, yet little is known about what happens to these stimulatory molecules within animal tissues. We tracked fluorescent and radiolabeled LPS from a s.c. inoculation site to its draining lymph nodes (DLN), blood, and liver. Although we found FITC-labeled LPS in DLN within minutes of injection, drainage of radiolabeled LPS continued for >6 wk. Within the DLN, most of the LPS was found in the subcapsular sinus or medulla, near or within lymphatic endothelial cells and CD169(+) macrophages. Whereas most of the LPS seemed to pass through the DLN without entering B cell follicles, by 24 h after injection a small amount of LPS was found in the paracortex. In wild-type mice, ≥70% of the injected radiolabeled LPS underwent inactivation by deacylation before it left the footpad; in animals that lacked acyloxyacyl hydrolase, the LPS-deacylating enzyme, prolonged drainage of fully acylated (active) LPS boosted polyclonal IgM and IgG3 Ab titers. LPS egress from a s.c. injection site thus occurred during many weeks and was mainly via lymphatic channels. Its immunological potency, as measured by its ability to stimulate polyclonal Ab production, was greatly influenced by the kinetics of both lymphatic drainage and enzymatic inactivation.     

Semisynthetic preparation of choline and ethanolamine plasmalogens

Choline and ethanolamine plasmalogens containing defined acyl chains are prepared by deacylation and reacylation of beef heart plasmalogens. During the reactions, the amino group of ethanolamine plasmalogens is protected by the trityl group. Deacylation is achieved by mild alkaline hydrolysis, and the lysoplasmalogens are reacylated with oleoylimidazolide in the presence of the methylsulfinylmethide anion. The protective group is removed from N-trityl ethanolamine plasmalogen by treatment with silicic acid in hexane. The choline and ethanolamine plasmalogens prepared by the procedures described are free of geometric, positional and steric isomers.     

Purification of acyloxyacyl hydrolase, a leukocyte enzyme that removes secondary acyl chains from bacterial lipopolysaccharides

Leukocytes contain an enzyme that detoxifies bacterial lipopolysaccharides (also called endotoxins) by removing fatty acyl chains that are attached in acyloxy acyl linkage to the glucosamine backbone of lipid A. We describe the purification of an enzyme that carries out this activity, termed acyloxyacyl hydrolysis, from the HL-60 human promyelocyte cell line. The enzyme is a glycoprotein of apparent Mr = 52,000-60,000 that is found in low abundance (less than 0.001% of the cell lysate protein), principally in the granule fraction of the cells. The protein has two disulfide-linked subunits of apparent Mr = 50,000 and 14,000-20,000, each of which contains N-linked oligosaccharides. This is the first lipopolysaccharide-degrading enzyme that has been purified from animal cells.     

Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils

Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil‐rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram‐negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild‐type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule‐localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation.     

Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei

Inositol acylation is an obligatory step in glycosylphosphatidylinositol (GPI) biosynthesis whereas mature GPI anchors often lack this modification. The GPI anchors of Trypanosoma brucei variant surface glycoproteins (VSGs) undergo rounds of inositol acylation and deacylation during GPI biosynthesis and the deacylation reactions are inhibited by diisopropylfluorophosphate (DFP). Inositol deacylase was affinity labelled with [3H]DFP and purified. Peptide sequencing was used to clone GPIdeAc, which encodes a protein with significant sequence and hydropathy similarity to mammalian acyloxyacyl hydrolase, an enzyme that removes fatty acids from bacterial lipopolysaccharide. Both contain a signal sequence followed by a saposin domain and a GDSL-lipase domain. GPIdeAc(-/-) trypanosomes were viable in vitro and in animals. Affinity-purified HA-tagged GPIdeAc was shown to have inositol deacylase activity. However, total inositol deacylase activity was only reduced in GPIdeAc(-/-) trypanosomes and the VSG GPI anchor was indistinguishable from wild type. These results suggest that there is redundancy in T.brucei inositol deacylase activity and that there is another enzyme whose sequence is not recognizably related to GPIdeAc.     

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.     

Deciphering the unusual acylation pattern of Helicobacter pylori lipid A

The synthesis of “typical” hexa-acylated lipid A occurs via a nine-step enzymatic pathway, which is generally well conserved throughout all gram-negative bacteria. One exception to the rule is Helicobacter pylori, which has only eight homologs to the nine lipid A biosynthetic enzymes. The discrepancy occurs toward the end of the pathway, with H. pylori containing only a single putative secondary acyltransferase encoded by jhp0265. In Escherichia coli K-12, two late acyltransferases, termed LpxL and LpxM, are required for the biosynthesis of hexa-acylated lipid A. Detailed biochemical and genetic analyses reveal that H. pylori Jhp0265 (the protein encoded by jhp0265) is in fact an LpxL homolog, capable of transferring a stearoyl group to the hydroxyl group of the 2′ linked fatty acyl chain of lipid A. Despite the lack of a homolog to LpxM in the H. pylori genome, the organism synthesizes a hexa-acylated lipid A species, suggesting that an equivalent enzyme exists. Using radiolabeled lipid A substrates and acyl-acyl carrier protein as the fatty acyl donor, we were able to confirm the presence of a second H. pylori late acyl transferase by biochemical assays. After synthesis of the hexa-acylated lipid A species, several modification enzymes then function to produce the major lipid A species of H. pylori that is tetra-acylated. Jhp0634 was identified as an outer membrane deacylase that removes the 3′-linked acyl chains of H. pylori lipid A. Together, this work elucidates the biochemical machinery required for the acylation and deacylation of the lipid A domain of H. pylori lipopolysaccharide.     

Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils

The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.     

MD-2: the Toll 'gatekeeper' in endotoxin signalling

Lipopolysaccharide (LPS) from the outer cell wall of Gram-negative bacteria is a potent stimulator of the mammalian innate immune system. The Toll-like receptor 4 (TLR4) pathway triggers the inflammatory responses induced by LPS in a process that requires the interaction of LPS-bound myeloid differentiation-2 (MD-2) with TLR4. Here we propose two possible mechanisms for LPS recognition and signalling that take into account both the structural information available for TLR4 and MD-2, and the determinants of endotoxicity, namely, the acylation and phosphorylation patterns of LPS. In our first model, LPS induces the association of two TLR4-MD-2 heterodimers by binding to two different molecules of MD-2 through the acyl chains of lipid A. In our second model, the binding of LPS to a single TLR4-MD-2 complex facilitates the recruitment of a second TLR4-MD-2 heterodimer. These models contrast with the activation of Drosophila Toll, where the receptor is crosslinked by a dimeric protein ligand.     

Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues.

The chemical structure of the free lipid A isolated from Mesorhizobium huakuii IFO 15243(T) was elucidated. Lipid A is a mixture of at least six species of molecules whose structures differ both in the phosphorylation of sugar backbone and in fatty acylation. The backbone consists of a beta (1'-->6) linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide that is partly substituted by phosphate at position 4'. The aglycon of the DAG-disaccharide has been identified as alpha-D-galacturonic acid. All lipid A species carry four amide-linked 3-hydroxyl fatty residues. Two of them have short hydrocarbon chains (i.e. 3-OH-i-13:0) while the other two have longer ones (i.e. 3-OH-20:0). Distribution of 3-hydroxyl fatty acids between the reducing and nonreducing DAG is symmetrical. The nonpolar as well as (omega-1) hydroxyl long chain fatty acids are components of acyloxyacyl moieties. Two acyloxyacyl residues occur exclusively in the nonreducing moiety of the sugar backbone but their distribution has not been established yet. The distal DAG amide-bound fatty acid hydroxyls are not stoichiometrically substituted by ester-linked acyl components.     

Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase

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>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>Eagg, Eagg:LBP (E/LBP approximately 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.     

Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone alpha-D-GalpA-(1-->4)-beta-D-DAG-(1-->6)-alpha-D-DAG-(1-->1)-alpha-D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[Delta(5)-12:1(3-OH)]] and 12:0[3-O(Delta(5)-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3-OH) and 12:0[3,6-(OH)( 2)] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF-alpha inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.     

Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells

Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14- tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-[14C]eicosatrienoate was two-to threefold that of 5,11,14-[14C]eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.