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The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-d-manno-octulosonic acid (Kdo)2-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo2-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and 1H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo2-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >103 in cells from TLR-4-deficient mice. The purity of Kdo2-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.
). These amphipathic substances not only are structural components of biological membranes but also play important roles in the pathophysiology of inflammation, atherosclerosis, and growth control. Additional lipid functions should emerge from the comprehensive analysis of macrophage lipids. Electrospray ionization/mass spectrometry (ESI/MS) (
), coupled with prefractionation methods like reverse-phase liquid chromatography (LC), is being applied systematically to set the stage for the seamless integration of lipid metabolism into the broader fields of genomics, proteomics, and systems biology. To facilitate this endeavor, LIPID MAPS has introduced a new comprehensive classification system for biological lipids, amenable to computer-based data processing and substructure comparison (
). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids, and 8) polyketides. More details are available on the LIPID MAPS web site (www.lipidmaps.org).
As an initial test of the LIPID MAPS approach, the time-dependent response of the macrophage to stimulation by lipopolysaccharide (LPS) is being investigated. LPS is a potent activator of the innate immunity receptor TLR-4/MD2 (
). It stimulates macrophages via its lipid anchor, which is termed lipid A (or endotoxin). Animal cells can detect picomolar lipid A concentrations using TLR-4/MD2 and accessory proteins such as CD14 and LPS binding protein (
). Excessive endotoxin exposure during severe Gram-negative sepsis contributes to shock, multiple organ failure, and death. A promising approach to the amelioration of endotoxin-induced illnesses has emerged with the discovery that certain synthetic lipid A analogs (
A limitation of using native LPS from wild-type Gram-negative bacteria for clinical or biological studies of endotoxin activity is its large size and micro-heterogeneity, especially in the length and composition of its terminal glycan chains (
). Direct detection and quantification by mass spectrometry of intact LPS in blood or biological samples is not yet possible. Accordingly, LPS levels are usually estimated by indirect methods, such as the clotting of the amoebocyte limulus lysate induced by the lipid A moiety of LPS (
). The tissue distribution and metabolism of LPS injected into animals have likewise been difficult to evaluate because of the same micro-heterogeneity problem. Knowledge of endotoxin tissue levels and metabolism might suggest new therapeutic approaches to problems of sepsis and inflammation.
We now report the large-scale purification, structural analysis, and biological characterization of a chemically defined LPS, consisting of lipid A and an attached 3-deoxy-d-manno-octulosonic acid (Kdo) disaccharide (Fig. 1, compound A) (
). This substance, designated Kdo2-Lipid A, was purified from an Escherichia coli K-12 mutant that synthesizes a truncated LPS because of a mutation in the enzymes that normally attach the heptose residues of the LPS core domain (
Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.
). This LPS preparation has an intact lipid A anchor, which is fully active as an endotoxin by various biological criteria, such as the stimulation of RAW 264.7 macrophage-like tumor cells to produce eicosanoids and tumor necrosis factor-α (TNFα). Kdo2-Lipid A is highly selective for TLR-4 and has the distinct advantage that it can be quantified by ESI/MS. The chemical purity of Kdo2-Lipid A is sufficient to enable high-resolution structural studies, such as NMR spectroscopy or X-ray crystallography of its complexes with important receptor proteins or enzymes (
Fig. 1Structure of 3-deoxy-d-manno-octulosonic acid (Kdo)2-Lipid A and related minor lipids purified from Escherichia coli WBB06. This mutant is defective in the WaaC and WaaF glycosyltransferases that attach heptose to the lipopolysaccharide (LPS) core (
The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives.
Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.
), compound F. Following purification, compound A represents ∼91%, compound B ∼5%, and compound F ∼3% of the total. B and F are expected have the same biological activity as A, given their similar structures (
Function of the Escherichia coli msbB gene, a multicopy suppressor of htrB Knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate incorporation by HtrB.
). The origin of F is unknown. Other molecular species still present as trace contaminants are compounds C, D, and E, each of which represents less than 1% of the total. The hepta-acylated derivative C arises from A in cells by the action of the PagP acyltransferase (
). Compounds D and E are decomposition products. The numbers of carbon atoms in each acyl chain are indicated. Key structural differences in compounds B through F are highlighted in red.
Tryptone, yeast extract, L-broth, tetracycline, and glucose were obtained from Difco. Chloroform and methanol were purchased from EMS. Cyclohexane was obtained from Burdick and Jackson. EDTA, t-butanol, and t-butyl ammonium phosphate (TBAP) were from Aldrich. Ammonium acetate and acetonitrile were obtained from Acros. Ethanol, ammonium hydroxide, isopropanol, and sodium chloride were from Fisher Scientific (Fair Lawn, NJ). Silica gel was provided by Grace Davison. Silica gel modified by Astec was used as the C18 reverse-phase resin. Whatman DE 23 DEAE cellulose ion exchange resin and Whatman Partsil K6 TLC plates were purchased from VWR. Lipid visualization after TLC was performed by spraying the plates with 10% H2SO4 in ethanol, followed by charring.
RAW 264.7 cells were obtained from the American Type Culture Collection. They were grown for no more than 24 passages on DMEM (Cellgro), supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Invitrogen; Carlsbad, CA). For cell stimulation experiments, the LPS from E. coli 0111:B4 was obtained from Sigma (# L4130). Kdo2-Lipid A was prepared as described below. All liquid chromatography/mass spectrometry (LC/MS) solvents used for prostaglandin analysis were OmniSolv grade or the equivalent from EMD Chemicals (Gibbstown, NJ). The formic acid was purchased from Fisher Scientific. All other fine chemicals were analytical reagent grade or better.
Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.
Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
). A 500 l capacity IF-500 fermentor was filled with 375 l of water, containing 3,750 g of tryptone (10 g/l), 1,875 g yeast extract (5 g/l), 3,750 g NaCl (10 g/l), and 750 g glucose (2 g/l). After sterilization, 4.5 g of tetracycline (12 mg/l) was added from a concentrated filter-sterilized stock solution in 70% aqueous ethanol. The pH of the growth medium was adjusted to 7.2 with NaOH and H3PO4. After 24 h of growth to early stationary phase at 37°C, the WBB06 cells were harvested by centrifugation in a CEPA Z-41 continuous-flow centrifuge. The cell paste (approximately 2 kg) was frozen at −80°C.
Extraction and purification of Kdo2-Lipid A from E. coli WBB06
The overall scheme for the purification is summarized in Fig. 2. A modified Folch extraction procedure was used to recover the lipids (
). The frozen WBB06 cells were dispersed in a single-phase solvent mixture consisting of 15 ml chloroform-methanol (2:1; v/v) per gram of cell paste. The suspended paste was stirred overnight at room temperature. The insoluble residue was removed, and the supernatant, containing the extracted lipids, was stored at −20°C. The residue was reextracted overnight at room temperature with 7.5 ml of chloroform-methanol (2:1; v/v) per gram of initial cell paste. The reextracted residue was discarded, and the second supernatant was pooled with the first. The combined supernatants were converted to a two-phase system by adding 20% of their total volume as aqueous 1.0 M NaCl. The lower organic phase was recovered, and the solvent was removed by rotary evaporation. The weight of the extracted lipids, which consist mainly of phosphatidyl-ethanolamine, phosphatidylglycerol, and Kdo2-Lipid A, was approximately 65.3 g, or 3.2% of the initial cell paste weight. The extracted lipids were stored neat at −20°C.
Fig. 2Electrospray ionization/mass spectrometry (ESI/MS) analysis in the negative mode of purified Kdo2-Lipid A. The sample at 10 μg/ml in acetonitrlle-water (70:30; v/v) containing 10 mM ammonium acetate was infused at 10 μl/min and scanned in the negative-ion mode in the Q3 of the API 4000 Q Trap® mass spectrometer. Uppercase letters A–F refer to the structures shown in Fig. 1. The m/z values for the major isotopic species are indicated.
The Kdo2-Lipid A was purified from the WBB06 membrane phospholipids on a normal-phase silica column, consisting of 20 g silica gel per gram total lipid. The silica was prepared by washing with two column volumes of 2% ammonium EDTA in methanol-water (1:1; v/v), followed by extensive washing with methanol-water (1:1; v/v) until the eluant pH was less than 8. The column was then washed with methanol, followed by chloroform. The extracted lipids were dissolved and loaded onto the column in chloroform-methanol (8:2; v/v). The lipids were eluted with a gradient that started at chloroform-methanol-water, 80:20:2 (v/v/v) and ended at 65:35:8 (v/v/v). Fractions containing Kdo2-Lipid A were identified by TLC (Rf, ∼0.3) using the mobile-phase chloroform-methanol-water (65:35:8; v/v/v). The fractions were pooled, and solvent was removed by rotary evaporation. The weight of lipids in the pooled fractions was 9.0 g, or 15% of the total lipid weight loaded onto the column. The Kdo2-Lipid A was ∼70% pure at this stage, as judged by HPLC (see below).
Purification by reverse-phase chromatography was performed as described previously on a C18 column to separate the predominant hexa-acylated Kdo2-Lipid A (Fig. 1) from minor hepta-, penta- and tetra-acylated lipid A species, and from minor phospholipid contaminants, using a two-solvent mixture. Solvent A is acetonitrile-water (1:1) with 1 mM TBAP, and solvent B consists of isopropanol-water (85:15; v/v) with 1 mM TBAP in water (
Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
). The reverse-phase chromatography column consisted of 60 g of resin per gram of lipid. The pooled fractions, containing the predominant hexa-acylated form of Kdo2-Lipid A, were extracted with chloroform and aqueous 0.5 M ammonium acetate. Solvent was removed by rotary evaporation, and the lipid was stored neat at −20°C.
Removal of TBAP and remaining pigments was accomplished by column chromatography on DEAE-cellulose in the solvent chloroform-methanol-water (2:3:1; v/v/v) with ammonium acetate elution (
Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
). The DEAE-cellulose column consisted of 30 ml DEAE cellulose bed volume per gram of lipid. Pooled fractions containing the Kdo2-Lipid A were extracted with chloroform and aqueous 0.5 M ammonium acetate. Solvent was removed by rotary evaporation, and the lipids were stored neat at −20°C.
Final purification to remove residual contaminants was achieved with a second normal-phase chromatography column, consisting of 20 g of silica gel per gram of lipid. Kdo2-Lipid A dissolved in chloroform-methanol was applied to the column, which was eluted as described above. Solvent was removed by rotary evaporation from the final pooled fractions containing the Kdo2-Lipid A. The residue was reconstituted in cyclohexane-t-butanol (1:1;v/v) and freeze dried. The final overall yield obtained by this procedure was 1.4 g of hexa-acylated Kdo2-Lipid A as the ammonium salt (>95% pure by HPLC). Long-term storage at −16°C to −24°C is recommended.
The Kdo2-Lipid A in the ampoules was prepared for use in cell stimulation experiments by dispersing it in sterile Dulbecco’s phosphate-buffered saline (DPBS) at 1 mg/ml by sonic irradiation in a bath sonicator for 5 min. A 100 μl portion of this stock was then diluted with 900 μl sterile DPBS. This 100 μg/ml (1,000×) aqueous dispersion of Kdo2-Lipid A was subjected to a second sonic irradiation prior to being added to the cells to yield a final concentration of 100 ng/ml in the growth medium.
HPLC analysis of Kdo2-Lipid A
HPLC analysis of Kdo2-Lipid A was performed using an Agilent 1100 quaternary pump system, equipped with a temperature-controlled column compartment and an in-line solvent degassing unit. The Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μ particle size) (Agilent Technologies; Palo Alto, CA) was maintained at 40°C. Samples were eluted using a gradient that consisted of methanol-water-chloroform (62:36:2; v/v/v) with 10 mM ammonium acetate as mobile-phase A and chloroform-methanol-water (80:20:2; v/v/v) with 50 mM ammonium acetate as mobile-phase B. The flow rate was 1 ml/min. Chemicals and solvents were HPLC grade and purchased from Fisher Scientific. The initial solvent, consisting of 85% A and 15% B, was maintained for 2 min, followed by a linear gradient to a final composition of 70% A and 30% B after 20 min; the solvent was held at the same composition until 30 min. A 10 min reequilibration of the column with 85% A and 15% B was performed prior to the next injection.
The material eluting from the column was directed to a SEDEX model 75C evaporative light-scattering detector (S.E.D.E.R.E., France). Zero-grade compressed air was used to nebulize the postcolumn flow stream at 3.5 bar into the detector at 50°C, set at a photomultiplier gain of 6. The detector signal was transferred to the Agilent HPLC Chemstation software for integration. The Kdo2-Lipid A samples were prepared at 1 mg/ml in a mixture of 85% A and 15% B (as defined above). Ten micrograms was injected onto the column for purity assessment by HPLC and evaporative light-scattering detection (ELSD).
Mass spectrometry of Kdo2-Lipid A and eicosanoids
Flow injection analysis of Kdo2-Lipid A was carried out using an ABI 4000 Q Trap® tandem quadrupole mass spectrometer (Applied Biosystems; Thornhill, Ontario, Canada) with negative-ion electrospray ionization during infusion of 10 μl/min of acetonitrile-water (70:30; v/v) containing 10 mM of ammonium acetate. The electrospray voltage was set at −4.5 kV, and nitrogen was used as curtain gas. LC/MS and low-resolution LC/MS/MS were carried out using a reverse-phase C8 HPLC column (Agilent), operated at a flow rate of 1 ml/min under gradient conditions, as described above. At this flow rate, the turbo ion spray interface was set at 600°C. For MS/MS experiments, nitrogen was used as collision gas at a collision energy of −15 V (laboratory frame of reference).
High-resolution electrospray mass spectrometry was carried out using a tandem hybrid quadrupole time-of-flight mass spectrometer (ABI Qstar XL, Applied Biosystems) by infusion of 50 ng/μl Kdo2-Lipid A in chloroform-methanol (2:1; v/v) at 6 μl/min. The instrument was scanned from m/z 100–2,500 in a negative-ion mode with the electrospray set at −4.2 kV. Nitrogen was used as collision gas and collisional activation of ions was performed at −40.0 V (laboratory frame of reference). This instrument was calibrated using polypropylene glycol 300 (Applied Biosystems) and had an average mass accuracy of 10–20 ppm.
The quantitative analysis of eicosanoids was carried out by LC/MS/MS using a tandem quadrupole mass spectrometer (ABI 4000 Q Trap®, Applied Biosystems), using stable isotope dilution and the multiple-reaction monitoring (MRM) mode. For each sample, a deuterium-labeled internal standard consisting of d4-PGE2 (99 atom % d4; Cayman Chemical, Ann Arbor, MI) was added to the cell supernatant just prior to sample workup. Cellular debris was removed by centrifugation (3,000 g, 10 min). Methanol and acetic acid were added to a final concentration of 10% and 2%, respectively. Eicosanoids were extracted using solid-phase extraction columns (Strata-X; Phenomenex, Rockville, IL). These columns were preconditioned by washing with 2 ml methanol, followed by 2 ml water. The sample was then loaded, and the column washed with 2 ml 0.5% methanol. The eicosanoids were eluted from the column using 1 ml of methanol. This eluate was dried under vacuum and redissolved in 100 μl of solvent system C [water-acetonitrile-formic acid (63:37:0.02; v/v/v)]. Standard curves were prepared in separate experiments in order to relate the abundance of MRM transitions to that of the deuterium-labeled internal standard (10 ng), using primary standards of each eicosanoid (Cayman Chemical).
Eicosanoids were separated by reverse-phase HPLC on a reverse-phase C18 column (2.1 mm × 250 mm; Grace-Vydac, Hesperia, CA) operated at a flow rate of 300 μl/min at 25°C. The column was initially equilibrated with 100% solvent C. Samples were then loaded through a 5 μl injection loop and eluted with a linear gradient from 0%–20% solvent D, consisting of acetonitrile-isopropanol (1:1; v/v), in 6 min; next, solvent D was increased to 55% in 6.5 min, and held until 10 min. Finally, solvent system D was linearly increased to 100% in 2 min and held for an additional 1 min. In order to recycle the column, the solvent system was switched to 100% C at 13.5 min and held until 16 min. The mass spectrometer was run in the MRM mode with the electrospray ion source operating in the negative mode (−4.5 kV), with the turbo ion spray source set at 525°C. Collisional activation of each of the eicosanoid precursor ions was carried out using nitrogen as collision gas. Specific MRM pairs are as follows: 11β-PGF2α and PGF2α at m/z 353 → 193 (−80 V); PGE2 and PDG2 at m/z 351 → 189 (−50 V); PGJ2 at m/z 333 → 189 (−30 V); 15D-PGD2 at m/z 333 → 271 (−30 V); 15D-PGJ2 at m/z 315 → 271 (−108 V); and 11-HETE at m/z 319 → 167 (−30 V). The deuterium-labeled internal standards (d4-PGE2 and d4-PGD2) were monitored at m/z 355 → 193. In some cases, the same MRM pairs were used to monitor different eicosanoids (i.e., PGE2 and PGD2); however, because these eluted at different HPLC retention times, they can be analyzed independently.
NMR spectroscopy of Kdo2-Lipid A
For NMR analysis, a 1 mg sample of Kdo2-Lipid A was dissolved in 0.3 ml CDCl3-CD3OD-D2O (2:3:1; v/v/v) and analyzed by 1H-COSY, TOCSY, and NOESY at 600 MHz, as described previously for lipid A and related compounds (
Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.
RAW 264.7 mouse macrophage tumor cells were plated in DMEM with 10% fetal bovine serum (HyClone Labs; Provo Utah), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Prior to stimulation, cells were plated at a density of 5 ×105 cells per well in 25 cm2 flasks and were allowed to adhere for 24 h. They were then washed with PBS and switched to fresh DMEM containing 0.5% fetal bovine serum for another 18 h. Cells were then exposed to either 100 ng/ml LPS (Sigma L4130 from E. coli 0111:B4) or 100 ng/ml of Kdo2-Lipid A for 24 h. Both the LPS and the Kdo2-Lipid A were suspended in PBS, and the solutions were bath-sonicated before each experiment.
TNF determination
TNFα levels were determined with the mouse TNFα immunoassay kit from R and D Systems, Inc. (Minneapolis, MN), according to the manufacturer’s instructions. Typically, 50 μl portions of the cell culture medium were removed from control and treated cells, and frozen at −20°C prior to assay. TNFα levels are expressed as pg/ml or ng/106 cells.
TLR-4 selectivity of Kdo2-Lipid A
Total bone marrow cells were isolated from C57Bl/6 (wild-type) and TLR4−/− mice (
), and then cultured in DMEM supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, and 30% L929 conditioned medium. Cells were seeded at 5 × 105/ml in DMEM, supplemented as above, and allowed to adhere for 2–3 h at 37°C in an atmosphere of 10% CO2. The cells were then washed with DMEM and supplemented with 10% serum and 1% penicillin-streptomycin, prior to 24 h stimulation with the indicated concentrations of Kdo2-Lipid A or the TLR2 ligand tripalmitoyl-S-glyceryl-cysteine (Pam3CYS). The concentrations of IL-6 in the culture supernatants were measured by ELISA, using reagents purchased from Pharmingen. The TLR4−/− mice were obtained from Dr. S. Akira (
RAW 264.7 mouse macrophage tumor cells were treated with 100 ng/ml LPS or Kdo2-Lipid A, prepared as described above, for 6 h. Total RNA was purified using RNeasy columns (Qiagen).
RNA analysis by Northern blotting was performed as described previously (
). Ten micrograms total RNA were separated by gel electrophoresis and transferred to nylon (Supercharge, Scheicher and Schuell). Prior to hybridization, membranes were cross-linked by UV treatment (Stratagene) and stained with Methylene Blue (Molecular Research Center, Cincinnati, OH). COX-2 and GAPDH probes (
) were generated by RT-PCR, followed by random prime labeling (Amersham Biosciences) and hybridization with QuikHyb (Stratagene). Microarray experiments were performed using the Codelink Uniset 1 mouse microarray platform (Amersham), according to the manufacturer's directions.
RESULTS
Extraction and purification of Kdo2-Lipid A from E. coli WBB06
) was used to recover the lipids from the cell paste. The total lipid extract was fractionated by normal-phase silica chromatography, yielding a mixture of major and minor Kdo2-Lipid A species (Fig. 1). The purity at this stage was estimated by HPLC to be ∼70% hexa-acylated Kdo2-Lipid A. Subsequent separation of the hexa-acylated material from the other molecular species was achieved by reverse-phase chromatography, followed by DEAE cellulose chromatography to remove TBAP and remaining pigments (
Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
). This step yielded the ammonium salt of Kdo2-Lipid A. Finally, a second normal-phase silica chromatography was used to remove residues carried over from the reverse-phase and DEAE resins. The overall yield from 2 kg of cell paste was 1.4 g of the ammonium salt of hexa-acylated Kdo2-Lipid A.
ESI/MS and HPLC analysis of purified Kdo2-Lipid A
A 1 mg/ml sample of the purified material was dissolved in chloroform-methanol-water (80:20:2; v/v/v), further diluted to 10 μg/ml with acetonitrile-water (70:30: v/v) containing 10 mM ammonium acetate, and infused into an ABI 4000 Q Trap® mass spectrometer at a flow rate of 10 μl/min. The mass range of m/z 500–1,400 was scanned in the negative-ion mode (Fig. 2). The most prominent species were interpreted as the [M-2H]2− ions of the hexa-acylated compounds A and B (Fig. 1) at m/z 1,118.2 and 1,104.2, respectively. These substances differ in the lengths of their 3′ secondary acyl chains, which are C14 for A and C12 for B (Fig. 1). Sodium and potassium addition adducts of A were observed at m/z 1,129.2 and 1,137.2, respectively. Minor impurities were seen at m/z 1,237.3 (compound C), m/z 1,004.2 (compound D), m/z 900.0 (compound E), and m/z 1,158.2 and 1,168.2 (compound F). The masses of labeled peaks shown in Fig. 2 represent the most abundant isotopic forms of each compound (Fig. 1), which are ∼1 amu larger than the exact masses due to the more abundant M+1 isotope (data not shown).
The Kdo2-Lipid A preparation was further evaluated for purity by HPLC/ELSD. A 10 μg sample was injected onto a reverse-phase column, as described above. The peak areas determined by this analysis more closely reflect the mole ratios of the various components than do the peak intensities observed by mass spectrometry (Fig. 2). However, neither method is quantitative when implemented without standards. The major peak at 21.45 min in the HPLC/ELSD experiment (Fig. 3A), representing compounds A and F (Fig. 1), was ∼94% of the total integrated area. The smaller peak at 20.97 min (Fig. 3A) is due to compound B, which is slightly less hydrophobic because laurate replaces a myristate chain in the left glucosamine unit of B (Fig. 1). The combined hexa-acylated species (i.e., compounds A, B, and F) represent 99.1% of the total area in the HPLC/ELSD analysis.
Fig. 3HPLC/evaporative light-scattering detection and liquid chromatography/mass spectrometry (LC/MS) analysis of purified Kdo2-Lipid A. A 10 μg sample injected onto a C8 reverse-phase column (A) shows a major component accounting for ∼94% of the total. This material corresponds to compound A and small amounts of F (as shown in Fig. 1). The chromatography was optimized with the sample dissolved in solvents A and B of the mobile phases (85:15; v/v). Other more or less polar combinations of these solvents resulted in sample precipitation on the column, peak splitting, poor resolution, and/or reduced sensitivity. Column loads above 50 μg resulted in peak broadening and splitting. Uppercase letters refer to the structures shown in Fig. 1. B: A 1 μg sample was injected onto a C8 reverse-phase column. Each tracing is the extracted ion chromatogram (XIC) from a scan of m/z 800–1,400 amu. Compound E (XIC = 899), 13.76 min; compound D (XIC = 1,104), 19.18 min; compound B (XIC = 1,004), 20.43 min; compound A (XIC = 1,118), 20.78 min; and compound C (XIC = 1,237), 23.49 min. Compound F (XIC = 1,158) elutes with compound A.
In a separate experiment, the LC system (Fig. 3A) was coupled to the mass spectrometer, and the effluent was scanned in the range of m/z 800–1,400 amu. The peaks detected in the direct infusion experiment (Fig. 2) were extracted from the total ion current with a mass offset of ±2 amu to cover the signals arising from all isotopic forms. In this case, a 1 μg sample was injected onto the column. The extracted ion chromatograms (XICs) for the major and minor peaks seen in Fig. 2 were overlaid in Fig. 3B to assess their order of elution in relation to the HPLC/ELSD analysis. The retention times for the compounds were comparable. Compound E (XIC = 899) elutes at 13.76 min, compound D (XIC = 1,004) at 19.18 min, compound B (XIC = 1,004) at 20.43 min, compound A (XIC = 1,118) at 20.78 min, and compound C (XIC = 1,237) at 23.49 min. Compound F (Fig. 1), which is the 1-diphosphate derivative of compound A (XIC = 1,158 for [M-2H]2−), coeluted with compound A at an approximate abundance of 2.8% relative to compound A (data not shown). This order of elution is consistent with the relative hydrophobicity of these substances (Fig. 1). Again, the relative intensity of each extracted ion peak cannot be correlated quantitatively to the relative molar abundance of each lipid species without the use of appropriate standards.
High-resolution ESI/MS, ESI/MS/MS, and 1H-NMR analysis
A high-resolution quadrupole time-of-flight tandem mass spectrometer (QSTAR®XL; Applied Biosystems) was used to evaluate the exact mass and collision-induced ion fragmentation pattern of the purified Kdo2-Lipid A. Consistent with the LC/MS and HPLC/ELSD analyses shown above, the predominant ions corresponded to a hexa-acylated Kdo2-Lipid A (Fig. 1, compound A). Under these conditions, the Kdo2-Lipid A forms both doubly and triply charged species, as judged by negative-ion ESI/MS. Figure 4Ashows the region of the spectrum corresponding to the major cluster of doubly charged ions derived from natural product, overlaid with the predicted isotopic distribution for a molecule with the molecular formula of compound A in Fig. 1 (predicted monoisotopic [M-2H]2− at m/z = 1,117.66). The observed m/z at 1,117.66 and the isotopic distribution match the predicted values exactly (Fig. 4A).
Fig. 4High-resolution ESI/MS and tandem ESI/MS/MS of the major hexa-acylated Kdo2-Lipid A molecular species. A: Simulation of the predicted peaks [M-2H]2− attributed to the major form of Kdo2-Lipid A (compound A in Fig 1). B: MS/MS analysis of m/z 1,117.7. For all labeled peaks, only the monoisotopic mass is given.
To analyze the structure in more detail, the doubly charged Kdo2-Lipid A ion (m/z =1,117.7) was subjected to collision-induced decomposition mass spectrometry. The resulting MS/MS spectrum is shown in Fig. 4B. All product ions in the MS/MS spectrum are consistent with the structure proposed for compound A in Fig. 1. The Kdo sugars are readily lost from the molecular ion, giving rise to the two major product ions at m/z 219.04 and 439.09. Lipid product ions corresponding to the loss of one or both Kdo residues are also observed. Loss of the Kdo disaccharide yields the doubly and singly charged product ions at m/z 897.6 and 1,796.16, respectively. Loss of one Kdo unit yields a doubly charged product ion at m/z 1,007.6. In addition to the loss of Kdo disaccharide, the neutral loss of the secondary myristate chain was observed, both as singly or doubly charged product ions at m/z 1,567.95 and 783.46, respectively. A more complete interpretation of the ESI/MS/MS spectrum of the Kdo2-Lipid A and its minor impurities is available on the LIPID MAPS website (www.lipidmaps.org) and should serve as a reference for the analysis of the structures of other lipid A preparations.
The 2D 1H-1H COSY of our Kdo2-Lipid A is shown in Fig. 5. It is consistent with previous in-depth studies of related saccharolipid molecules (
Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.
Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.
). Although many of the protons of the Kdo units are not resolved from each other, the spectrum nevertheless supports the proposed structure shown in Fig. 1A. The connectivity of the glucosamine ring protons is indicated in Fig. 5. The NMR analysis gives no evidence for other impurities that might have been overlooked by HPLC and ESI/MS.
Fig. 51H-NMR COSY of Kdo2-Lipid A purified from WBB06. Diagnostic cross-peaks arising from proton-proton couplings in the proximal and distal glucosamine residues are labeled according to the scheme shown in Fig. 1. The spectrum is in accord with previous studies of lipid A NMR spectra (
Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.
LC/MS analysis of eicosanoids from stimulated RAW cells
In the course of setting up the LC/MS system for the comprehensive analysis of eicosanoids in mouse macrophages, we have established a library that includes chromatographic retention times and tandem mass spectrometry data for the most common eicosanoids. To date, this includes over 70 compounds, and we are aggressively updating and expanding its contents. This library is available to the scientific community on the website www.lipidmaps.org. The web visitor can obtain chemical structures for standards in both GIF and ChemDraw® formats, specific details regarding LC and MS parameters employed in our analysis, fragmentation spectra and structures of dominant fragment ions (including literature references, when available, for fragment assignments), and retention times for a stated set of chromatographic conditions. Finally, a web link to Cayman Chemical provides useful information and references on specific eicosanoids.
We have employed this procedure to compare the stimulation of RAW cells with both LPS or Kdo2-Lipid A. Addition of either ligand yielded the same eicosanoid profile (Fig. 6). The major eicosanoid produced was PGD2 (130 ng/106 cells). PGF2α and PGE2 were also present, but only at 2–5 ng/106 cells. In addition, significant amounts of the PGD2 breakdown products 11β-PGF2α, PGJ2, 15 deoxy PGD2, and 15 deoxy PGJ2 were detected (3–15 ng/106 cells). Trace amounts (less than 1 ng/106 cells) of 11 HETE and arachidonic acid were also routinely detected. The 11 HETE was found to be the 11 (R) HETE, as determined by chiral HPLC, demonstrating that it is a product of a COX enzyme rather than 5-lipoxygenase. In fact, significant levels of 5-lipoxygenase-derived products were not detected with this stimulation protocol. The baseline for eicosanoid production in untreated cells is zero.
Fig. 6Profile of prostaglandin production in Kdo2-Lipid A-stimulated RAW cells. RAW 264.7 cells were stimulated with 100 ng/ml of either LPS (open bars) or Kdo2-Lipid A (solid bars) for 24 h, as outlined in Experimental Procedures, and the eicosanoid levels were determined by LC/MS/MS. The LPS is very heterogeneous, and its average molecular weight is on the order of 10,000 Da. Error bars represent standard deviation of triplicate measurements.
We next determined the dose response of the RAW cells to these two ligands. For these studies, we report only the values for PGE2 and PGD2, and the cytokine TNFα, but all of the compounds identified in Fig. 6, within error, followed the same general pattern. Figure 7clearly shows that RAW cells exhibit similar responses to LPS or Kdo2-Lipid A for PGE2, PGD2, and TNFα. The maximum responses elicited for each type of saccharolipid were usually within experimental error. For the eicosanoids, the EC50 for the LPS was slightly higher than for Kdo2-Lipid A, perhaps because of the greater purity and smaller molecular mass of Kdo2-Lipid A. Taking PGD2 as an example, the EC50 for LPS was 79 ± 11 ng/ml, whereas for Kdo2-Lipid A, it was 11 ± 1 ng/ml. Unlike the eicosanoids, the EC50s for the TNFα response for each saccharolipid were within experimental error, i.e., LPS was 204 ± 22 ng/ml and Kdo2-Lipid A was 140 ± 70 ng/ml.
Fig. 7Dose response curves for Kdo2-Lipid A- or LPS-stimulated RAW cells. RAW 264.7 cells were stimulated for 24 h with either Kdo2-Lipid A (closed circles) or LPS (open circles), following the protocol outlined in Experimental Procedures. The release of PGD2 is shown in panel A, and that of PGE2 is shown in panel B. Panel C shows the release of tumor necrosis factor-α (TNFα). The lines were obtained by nonlinear regression analysis fitting the data to a four-parameter logistic equation based on a simple binding model (SigmaPlot 9.0, Systat Software, Point Richmond, CA). Error bars represent standard deviation of triplicate measurements.
The time courses of stimulation are shown in Fig. 8. The cells were exposed to either LPS or Kdo2-Lipid A, as indicated. After 30 min exposure to either ligand, the eicosanoid levels in the media were unchanged from the control levels. By 2 h, there was a 10-fold increase in the eicosanoids in the media from cells stimulated by either ligand. At 24 h, the fold activation of eicosanoid production over controls varied between 500 and several thousand. The TNFα response started sooner than that of the eicosanoids; the levels of TNFα at 2 h were often 1,000-fold higher than at 30 min. The maximum level reached at 24 h was 5,000-fold higher than at 30 min for LPS. The maximum response to LPS stimulation was usually 1.5 to 2 times that for Kdo2-Lipid A stimulation.
Fig. 8Time course of eicosanoid release following stimulation of RAW cells with either Kdo2-Lipid A or LPS. The RAW 264.7 cells were stimulated with 100 ng/ml of either Kdo2-Lipid A (closed circles) or LPS (open circles), as outlined in Experimental Procedures, with the exception that the exposure to ligand was varied from 0 to 24 h. The release of PGD2 is shown in panel A, that of PGE2 is shown in panel B, and that of TNFα is shown in panel C. The lines are best fits to the data points, but do not represent theoretical fits to any particular model. Error bars represent standard deviation of triplicate measurements.
The data presented here represent typical results obtained from at least three experiments carried out on different days and employing cells at different passages. All data points represent the average of at least triplicates run on the same day.
TLR-4 selectivity
As shown in Fig. 9, bone marrow macrophages from wild-type mice responded fully to 10 ng/ml Kdo2-Lipid A, whereas TLR4−/− macrophages did not. In fact, there was no measurable response above the buffer control in the TLR4−/− cells at 100 ng/ml Kdo2-Lipid A. Only at 1 μg/ml Kdo2-Lipid A was there a small response in the TLR4−/− cells, suggesting the presence of small amounts of a TLR2-activating contaminant, possibly a bacterial lipopeptide. Both the wild-type and the TLR4−/− bone marrow-derived macrophages also responded vigorously to a synthetic Pam3CYS standard, a well-characterized TLR2 ligand (data not shown) (
Fig. 9Toll-like receptor-4 selectivity of purified Kdo2-Lipid A in mouse bone marrow macrophages. WT, wild type. Error bars represent standard deviation.
To investigate the relative biological activities of LPS and Kdo2-Lipid A at the level of global gene expression, experiments were performed comparing the responses of RAW 264.7 cells to both agonists. Engagement of the TLR4 receptor results in the upregulation of hundreds of genes secondary to activation of numerous transcription factors, including NF-κB, AP-1, and IRF3 (
). RAW 264.7 cells were treated for 6 h with 100 ng/ml Kdo2-Lipid A, 100 ng/ml LPS, or control buffer. The total RNA was extracted and then analyzed on microarrays representing approximately 10,000 independent transcripts. The patterns of inducible genes following LPS or Kdo2-Lipid A treatment were highly concordant, as illustrated for the 200 most highly inducible LPS target genes (Fig. 10). These results were confirmed for the COX2 gene by Northern blotting (Fig. 10, inset). There were no examples of genes that were highly induced by Kdo2-Lipid A (i.e., >3-fold) but not induced by LPS. Only one gene in the top 200 genes that were highly induced by LPS was not induced by Kdo2-Lipid A. This gene corresponds to Mad, a transcription factor that operates as a negative regulator of Myc-dependent transcription (
). Its induction by LPS but not Kdo2-Lipid A in these studies may be due to contaminants in the LPS preparation that regulate gene expression by TLR4-independent mechanisms. These findings indicate that Kdo2-Lipid A is a functional mimetic of LPS at the level of gene expression for broad sets of TLR4-sensitive genes.
Fig. 10Comparison of the effects of LPS and Kdo2-Lipid A on gene expression in RAW 264.7 macrophages. RAW 264.7 cells were treated with 100 ng/ml LPS, 100 ng/ml Kdo2-Lipid A, or the control buffer. Total RNA was collected 6 h following treatment and used for microarray analysis on Codelink Mouse Uniset 1 microarrays. The fold induction of the 200 most highly LPS-responsive genes is compared with the fold induction of these genes in response to Kdo2-Lipid A. Inset: Northern blot analysis of total RNA (10 μg) isolated from control, LPS-treated or Kdo2-Lipid A-treated RAW 264.7 cells for COX2 and GAPDH.
) has intensified the search for chemically defined ligands to enable structural and mechanistic studies. The lipid A (endotoxin) moiety of enterobacterial LPS (
), but well-characterized synthetic or natural lipid A preparations are not generally available. Most studies of TLR-4 activation, including the recent comprehensive characterization of gene expression in humans following endotoxin infusion (
). Furthermore, most intact LPS preparations have not been characterized by mass spectrometry or NMR spectroscopy, primarily because of their heterogeneity, large size, and insolubility (
Distribution of lipid A species between long and short chain lipopolysaccharides isolated from Salmonella, Yersinia, and Escherichia as seen by 252Cf plasma desorption mass spectrometry.
, are not easy to prepare and are not readily available to the scientific community. Kdo2-Lipid A represents an even greater synthetic challenge than lipid A per se (
). However, the addition of phosphate groups and acyl chains to the glucosamine disaccharide backbone of lipid A presents special technical problems, and the glycosidic linkages formed by Kdo are relatively unstable in the presence of mild acid (
The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives.
). Chemo-enzymatic approaches, using synthetic lipid A in conjunction with recombinant, purified Kdo transferase, may offer the best long-term solution for the future. However, at the present time, the preparation of lipid A and related molecules from large-scale fermentations of selected Gram-negative bacteria remains the best option.
As part of the systematic LIPID MAPS effort to quantify all macrophage lipids in response to endotoxin stimulation, we have developed a new large-scale purification of Kdo2-Lipid A (Fig. 1, compound A), a substructure of LPS made by heptose-deficient mutants of E. coli (
). This material can be extracted from cells with chloroform-methanol mixtures, like a glycerophospholipid, i.e., without the use of phenol or mild-acid hydrolysis (
Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
). It can be purified on a large scale by a combination of silica, ion-exchange, and reverse-phase chromatography. As shown in Fig. 2, Fig. 3, Fig. 4, Fig. 5, intact Kdo2-Lipid A from E. coli WBB06 is amenable to full structural analysis by high-resolution ESI/MS and NMR methodology. It has demonstrated stability for 9 months as the ammonium salt, when stored neat in ampoules at −16°C to −24°C. Extended stability studies are ongoing. Over 99% of the purified material is hexa-acylated, with ∼91% as compound A, ∼5% as B, and 3% as F (Fig. 1, Fig. 2, Fig. 3).
) have recently detected trace amounts of E. coli Kdo2-Lipid A species that contain odd-chain fatty acids, which might arise by the priming of fatty acid biosynthesis with propionyl-CoA instead of acetyl-CoA (
). It is unlikely that these species or the other minor impurities detected (Figs. 2, 3) would have a significant impact on the biological activity of the major component, compound A (Fig. 1). In any event, these contaminants would also be present in all other E. coli or Salmonella LPS preparations.
The structural characterization of our Kdo2-Lipid A exceeds that of any previously reported natural lipid A or LPS. Its biological activity is comparable to that of our current lot of Sigma L4130 LPS (from E. coli 0111:B4) made by conventional procedures (Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10). The reproducibility and chemically defined nature of our Kdo2-Lipid A should make it very attractive as a standard for pharmaceutical quality control.
Although hexa-acylated E. coli lipid A by itself has significant biological activity, we chose to use Kdo2-Lipid A (also termed ReLPS) for our studies, because it is supposedly ∼10 times more potent than lipid A (
The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives.
Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.
). Treatment of LPS with mild acid introduces additional micro-heterogeneity into lipid A, such as the partial dephosphorylation of the 1 position or the elimination of an acyl chain (
). It is likely that Kdo2-Lipid A is slightly more active than lipid A because it is dispersed more efficiently in aqueous systems. The availability of large amounts of Kdo2-Lipid A should enable in-depth studies of lipid A physical properties in water, such as the determination of its critical micelle concentration, its phase behavior as a function of pH and various counter ions, and its formation of complexes with proteins. Such studies are underway (S. White, personal communication).
A significant advantage of Kdo2-Lipid A over LPS is that it is a reproducible, well-defined substance that should be detectable with great sensitivity by ESI/MS and/or MRM after uptake by cultured macrophages or injection into animals. This feature will permit quantitative pharmacokinetic and metabolic studies, which are not possible with full-length LPS. In this context, it would be of interest to compare Kdo2-Lipid A to LPS under the recently published human endotoxin infusion protocols (
), given that plasma, urine, and circulating white cell levels of Kdo2-Lipid A could be determined in parallel with markers of bioactivity. To facilitate quantification by mass spectrometry, it will be necessary to develop an additional external standard, such as a uniformly 13C-labeled Kdo2-Lipid A analog. Known amounts of this material would then be added to lipid mixtures extracted from plasma or tissues, and would allow for the quantification by mass spectrometry of any unlabeled Kdo2-Lipid A present in such samples. A 13C-labeled Kdo2-Lipid A standard could easily be prepared form WBB06 cells grown on minimal medium with [U-13C]glucose as their sole carbon source, using the purification protocols described above.
Kdo2-Lipid A is an excellent substrate for diverse lipid A modification enzymes. For instance, Kdo2-Lipid A is deacylated by PagL at the 3 position (
MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of francisella novicida LpxE expressed in Escherichia coli.
A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant.
An inner membrane enzyme in Salmonella typhimurium and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction in polymyxin-resistant mutants and role of a novel lipid-linked donor.
). These enzymes can act singly or in combination. The many resulting derivatives of Kdo2-Lipid A may prove useful as immuno-modulators, because some of these modifications are expected to alter the interactions of the lipid with TLR-4. For instance, the combined action of PagL and LpxE on Kdo2-Lipid A generates the same lipid A substructure currently used in the clinic as an adjuvant (
), but with the retention of the Kdo disaccharide. Our Kdo2-Lipid A therefore provides an ideal starting material for exploring these and other chemo-enzymatic transformations.
A major remaining challenge in elucidating the function of innate immunity receptors is the determination of their three-dimensional structures in the presence of bound ligands. X-ray structures of TLR-3 and of CD14, both without bound ligands, have recently been reported (
). A solution structure of MD2 with Kdo2-Lipid A might also be of considerable interest. We believe that our Kdo2-Lipid A is of sufficient purity to merit consideration as a ligand for cocrystallization trials. The possibility of generating pure 13C-labeled Kdo2-Lipid A, as described above, should greatly facilitate NMR solution studies of complexes with MD2, or CD14. Heptose-deficient “Re” LPS from Salmonella minnesota, which is often used to stimulate immune cells, would not be suitable for such biophysical studies, because of its considerable micro-heterogeneity with regard to the number of acyl chains and polar substituents (
Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination.
In summary, we have devised an efficient new purification scheme for obtaining large amounts Kdo2-Lipid A from E. coli and have demonstrated its functionality in activating macrophage genes, eicosanoids, and cytokines. We believe that our analytical technology, especially the HPLC system shown in Fig. 2, will be applicable to diverse lipid A molecules of natural and synthetic origin. Entirely different saccharolipids, such as mycobacterial trehalose derivatives (
), could also be purified on a large scale and analyzed by our procedures.
Acknowledgments
This work was supported by the LIPID MAPS Large Scale Collaborative, Grant GM-069338 from the National Institutes of Health. We are grateful to the National Institutes of General Medical Sciences for supporting this effort.
References
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Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.
Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.
Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.
Distribution of lipid A species between long and short chain lipopolysaccharides isolated from Salmonella, Yersinia, and Escherichia as seen by 252Cf plasma desorption mass spectrometry.
The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives.
Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.
MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of francisella novicida LpxE expressed in Escherichia coli.
A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant.
An inner membrane enzyme in Salmonella typhimurium and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction in polymyxin-resistant mutants and role of a novel lipid-linked donor.
Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination.
Function of the Escherichia coli msbB gene, a multicopy suppressor of htrB Knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate incorporation by HtrB.
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