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.     

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.     

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.     

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.     

Deacylation of lipopolysaccharide in whole Escherichia coli during destruction by cellular and extracellular components of a rabbit peritoneal inflammatory exudate

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.     

Biochemical transformation of bacterial lipopolysaccharides by acyloxyacyl hydrolase reduces host injury and promotes recovery

Animals can sense the presence of microbes in their tissues and mobilize their own defenses by recognizing and responding to conserved microbial structures (often called microbe-associated molecular patterns (MAMPs)). Successful host defenses may kill the invaders, yet the host animal may fail to restore homeostasis if the stimulatory microbial structures are not silenced. Although mice have many mechanisms for limiting their responses to lipopolysaccharide (LPS), a major Gram-negative bacterial MAMP, a highly conserved host lipase is required to extinguish LPS sensing in tissues and restore homeostasis. We review recent progress in understanding how this enzyme, acyloxyacyl hydrolase (AOAH), transforms LPS from stimulus to inhibitor, reduces tissue injury and death from infection, prevents prolonged post-infection immunosuppression, and keeps stimulatory LPS from entering the bloodstream. We also discuss how AOAH may increase sensitivity to pulmonary allergens. Better appreciation of how host enzymes modify LPS and other MAMPs may help prevent tissue injury and hasten recovery from infection.     

Transacylase formation of bis(monoacylglycerol)phosphate

Recent work within our laboratory has focused on the enzymes we hypothesize are involved in the biosynthesis of bis(monoacylglycerol)phosphate from phosphatidylglycerol. Here we describe a transacylase, active at acidic pH values, isolated from a macrophage-like cell line, RAW 264.7. This enzyme acylates the head group glycerol of sn-3:sn-1' lysophosphatidylglycerol to form sn-3:sn-1' bis(monoacylglycerol)phosphate. Here we demonstrate that this enzyme uses two lysophosphatidylglycerol molecules, one as an acyl donor and another as an acyl acceptor, and that the acyl contributions from all other lipids tested are comparatively minor. This enzyme prefers saturated acyl chains to monounsaturates, 16 and 18 carbon fatty acids over 14 carbon fatty acids, and saturated acyl chains at the sn-1 position to monounsaturated acyl chains on the sn-2 carbon of lysophosphatidylglycerol. We present data which show the transacylase activity depends on the presence of a lipid-water interface and the lipid polymorphic state.     

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.     

Acyl specificity of hamster heart CDP-choline 1,2-diacylglycerol phosphocholine transferase in phosphatidylcholine biosynthesis

The acyl specificity of 1,2-diacylglycerol: CDP-choline phosphocholine transferase (EC 2.7.8.2) for the formation of phosphatidylcholine with the appropriate acyl groups in hamster heart was investigated. Enzyme activity was determined in the microsomal fraction with 1,2-diacylglycerols of known acyl content. Maximum enzyme activity was obtained with diacylglycerol containing a monoenoic acyl group at the C-2 position of the glycerol moiety, regardless of the acyl group at the C-1 position. The specificity of the enzymes was also investigated by perfusing the isolated hamster heart with labelled glycerol. Comparison of the molecular species of the labelled diacylglycerols and phosphatidylcholine subsequent to perfusion revealed that the specificity of phosphocholine transferase was not limited to the monoenoic species of diacylglycerol. The difference in specificity observed between the in vitro assay and the perfusion study may partly be attributed to the presence of detergent in the enzyme assay mixture (to facilitate solubility of diacylglycerol). It is concluded that in the hamster heart, phosphocholine transferase has only limited ability to select the appropriate acyl groups for phosphatidylcholine biosynthesis. It appears that the majority of the newly formed phosphatidylcholine in the heart via the CDP-choline pathway is subsequently resynthesized by deacylation-reacylation process.     

Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.     

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Normal- and reverse-phase HPLC separations of fluorescent (NBD) lipids

We have developed two high-performance liquid chromatography methods for separating a number of fluorescent 4-nitrobenzo-2-oxa-1,3-diazole (NBD) analogs of glycerolipids and sphingolipids. Samples of fluorescent lipid analogs containing NBD-aminocaproyl (C6-NBD) or NBD-aminododecanoyl (C12-NBD) acyl chains were synthesized and analyzed by the following HPLC methods. An isocratic normal-phase method permitted resolution of a mixture of the 1,2-(palmitoyl, C6-NBD)-analogs of triacylglycerol, diacylglycerol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in less than 10 min, while a mixture of the (C6-NBD)-labeled analogs of ceramide, glucocerebroside, and sphingomyelin was separated in approximately 15 min. This method also detected various (C6-NBD)-phosphatidylcholine and -phosphatidylethanolamine molecules which differed only in their nonfluorescent acyl (oleoyl or palmitoyl) chains, and readily separated nonfluorescent dipalmitoylphosphatidylcholine from both (C6-NBD)- and (C12-NBD)-phosphatidylcholine derivatives. An isocratic reverse-phase system permitted separation of isomers of fluorescent phosphatidylcholine, -ethanolamine, -glycerol, -inositol, -serine, and phosphatidic acid in which the NBD-fatty acid was present in either the sn-1 or sn-2 position of the glycerol backbone.     

PAF-acetylhydrolases

Platelet-activating factor acetylhydrolases (PAF-AHs, EC 3.1.1.47) constitute a unique subfamily of phospholipases A(2), specific for short acyl chains in the sn-2 position of the phospholipid. Their primary substrate is the platelet-activating factor, PAF, from which they cleave an acetyl moiety with concomitant release of lysoPAF. However, some acetylhydrolase will also hydrolyze other polar phospholipids with up to 6-carbons long acyl chains in the sn-2 position. PAF-acetylhydrolases are diverse enzymes, and the well-characterized isoforms are serine-dependent hydrolases, which do not require Ca(2+) for activity. Given the existence of two pools of PAF, intra- and extracellular, the acetylhydrolases can be divided into two subclasses: those found in the cytosol and enzymes secreted to blood plasma or other body fluids. Recent crystallographic studies shed new light on the complex structure-function relationships in PAF-AHs.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence

The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. beta-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.     

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.     

Host inactivation of bacterial lipopolysaccharide prevents prolonged tolerance following gram-negative bacterial infection

A transient state of tolerance to microbial molecules accompanies many infectious diseases. Such tolerance is thought to minimize inflammation-induced injury, but it may also alter host defenses. Here we report that recovery from the tolerant state induced by Gram-negative bacteria is greatly delayed in mice that lack acyloxyacyl hydrolase (AOAH), a lipase that partially deacylates the bacterial cell-wall lipopolysaccharide (LPS). Whereas wild-type mice regained normal responsiveness within 14 days after they received an intraperitoneal injection of LPS or Gram-negative bacteria, AOAH-deficient mice had greatly reduced proinflammatory responses to a second LPS injection for at least 3 weeks. In contrast, LPS-primed Aoah- knockout mice maintained an anti-inflammatory response, evident from their plasma levels of interleukin-10 (IL-10). LPS-primed Aoah-knockout mice experiencing prolonged tolerance were highly susceptible to virulent E. coli challenge. Inactivating LPS, an immunostimulatory microbial molecule, is thus important for restoring effective host defenses following Gram-negative bacterial infection in animals.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Structural requirements for diacylglycerols to mimic tumor-promoting phobol diester action on the epidermal growth factor receptor

The structural requirements for diacylglycerols to mimic the action of tumor-promoting phorbol diesters on the epidermal growth factor (EGF) receptor of A431 human epidermoid carcinoma cells were investigated. Five biological effects were considered: inhibition of high affinity 125I-EGF binding, change in the phosphorylation state of the EGF receptor, inhibition of the EGF-dependent tyrosine phosphorylation of the EGF receptor, inhibition of [3H]phorbol 12 beta, 13 alpha-dibutyrate binding, and stimulation of calcium- and phospholipid-dependent protein kinase (C-kinase) in vitro. A marked effect of the acyl chain length, 3-10 carbons, of symmetric sn-1,2-diacylglycerols was observed on their ability to mimic the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). sn-1,2-Dipropanoylglycerol did not mimic the effects of PMA, but sn-1,2-didecanoylglycerol potently mimicked PMA action. A correlation was found between the ability of these diacylglycerols to stimulate the activity of C-kinase in vitro and to mimic the effects of PMA on the EGF receptor in intact cells. Analogues of sn-1,2-dioctanoylglycerol in which the 3' hydroxyl group was substituted with hydrogen, thio or chloro moieties were inactive when assayed for their ability to stimulate C-kinase in vitro and mimic PMA action in intact cells. We conclude that the hydroxyl group of a diacylglycerol is vital for the interaction with the phorbol diester receptor. The stringent correlation between the potency of the 11 diacylglycerol analogues tested to modulate C-kinase in vitro and to mimic PMA action in vivo provides strong evidence for the hypothesis that C-kinase plays a central role in the regulation of A431 cell EGF receptors by tumor-promoting phorbol diesters.