Kdo 2-Lipid A of Escherichia coli , a defined endotoxin that activates macrophages via TLR4

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 H-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 .10 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.—Raetz, C. R. H., T. A. Garrett, C. M. Reynolds, W. A. Shaw, J. D. Moore, D. C. Smith, Jr., A. A. Ribeiro, R. C. Murphy, R. J. Ulevitch, C. Fearns, D. Reichart, C. K. Glass, C. Benner, S. Subramaniam, R. Harkewicz, R. C. Bower-Gentry, M. W. Buczynski, J. A. Cooper, R. A. Deems, and E. A. Dennis. Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4. J. Lipid Res. 2006. 47: 1097–1111. Supplementary key words 3-deoxy-D-manno-octulosonic acid-Lipid A . Escherichia coli . Re . endotoxin . macrophage . mass spectrometry . LIPID MAPS . toll-like receptor-4 The LIPID MAPS consortium is developing quantitative methods for evaluating the composition, biosynthesis, and function of all macrophage lipids (1). 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) (2, 3), coupled with prefractionation methods like reversephase 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 (4). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids, Manuscript received 13 January 2006 and in revised form 7 February 2006. Published, JLR Papers in Press, February 14, 2006. DOI 10.1194/jlr.M600027-JLR200 Abbreviations: COX-2, cyclo-oxygenase 2; DPBS, Dulbecco’s phosphate-buffered saline; ELSD, evaporative light-scattering detection; ESI/ MS, electrospray ionization/mass spectrometry; Kdo, 3-deoxy-D-mannooctulosonic acid; LC/MS, liquid chromatography/mass spectrometry; LPS, lipopolysaccharide; MRM, multiple-reaction monitoring; Pam3CYS, tripalmitoyl-S -glyceryl-cysteine; TBAP, t-butyl ammonium phosphate; TLR, toll-like receptor; TNFa, tumor necrosis factor-a; XIC, extracted ion chromatogram. 1 To whom correspondence should be addressed. e-mail: edennis@ucsd.edu Copyright D 2006 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 47, 2006 1097 by gest, on D ecem er 8, 2017 w w w .j.org D ow nladed fom 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 timedependent 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 (5–7). LPS, a saccharolipid glycan according to the new LIPID MAPS classification scheme (4), is present in the outer membranes of most Gram-negative bacteria (6, 8–10). 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 (5, 7, 11, 12). Human volunteers injected with 4 ng/kg of LPS develop fever and a flu-like illness, lasting 24 to 48 h (13). Recent clinical studies in humans have characterized the complex response to endotoxin injection by using micro-array technology (14, 15). This approach revealed that the transcription of hundreds of genes is activated or repressed in human leukocytes following exposure to endotoxin (15). 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 (13, 16) or precursors (17) can antagonize the effects of lipid A (endotoxin) on TLR-4/MD2. The former are currently in clinical trials (13, 16). A limitation of using native LPS from wild-type Gramnegative 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 (6, 9). 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 (18, 19). 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-Dmanno-octulosonic acid (Kdo) disaccharide (Fig. 1, compound A) (6). 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 (6, 20, 21). 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-a (TNFa). 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 (7). EXPERIMENTAL PROCEDURES