Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.

TitleKdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.
Publication TypeJournal Article
Year of Publication2006
AuthorsRaetz CRH, Garrett TA, Reynolds MC, Shaw WA, Moore JD, Smith DC, Ribeiro AA, Murphy RC, Ulevitch RJ, Fearns C, Reichart D, Glass CK, Benner C, Subramaniam S, Harkewicz R, Bowers-Gentry RC, Buczynski MW, Cooper JA, Deems RA, Dennis EA
JournalJ Lipid Res
Volume47
Issue5
Pagination1097-111
Date Published2006 May
ISSN0022-2275
KeywordsAnimals, Chromatography, High Pressure Liquid, Escherichia coli, Lipopolysaccharides, Macrophage Activation, Mice, Nuclear Magnetic Resonance, Biomolecular, Prostaglandin D2, Spectrometry, Mass, Electrospray Ionization, Toll-Like Receptor 4
Abstract

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. Kdo(2)-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 (1)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. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.

DOI10.1194/jlr.M600027-JLR200
PubMed URLhttp://www.ncbi.nlm.nih.gov/pubmed/16479018?dopt=Abstract
Alternate TitleJ. Lipid Res.
PubMed ID16479018