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Journal of Lipid Research, Vol. 47, 1097-1111, May 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

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

Christian R. H. Raetz^*, Teresa A. Garrett^*, C. Michael Reynolds^*, Walter A. Shaw, Jeff D. Moore, Dale C. Smith, Jr., Anthony A. Ribeiro^*, Robert C. Murphy, Richard J. Ulevitch^**, Colleen Fearns^**, Donna Reichart, Christopher K. Glass, Chris Benner, Shankar Subramaniam, Richard Harkewicz^***, Rebecca C. Bowers-Gentry^***, Matthew W. Buczynski^***, Jennifer A. Cooper^***, Raymond A. Deems^*** and Edward A. Dennis^1,***

^* Department of Biochemistry, Duke University Medical Center, PO Box 3711, Durham, NC
Avanti Polar Lipids, Inc., 700 Industrial Park Drive, Alabaster, AL
Department of Pharmacology, University of Colorado Health Sciences Center, Mail Stop 8303, PO Box 6508, Aurora, CO
^** Department of Immunology, Scripps Institute, La Jolla, CA
Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
^*** Department of Chemistry and Biochemistry and Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA

Published, JLR Papers in Press, February 14, 2006.

¹ To whom correspondence should be addressed. e-mail: edennis{at}ucsd.edu

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.

Supplementary key words 3-deoxy-D-manno-octulosonic acid-Lipid A • Escherichia coli • Re • endotoxin • macrophage • mass spectrometry • LIPID MAPS • toll-like receptor-4

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-manno-octulosonic 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; TNF, tumor necrosis factor-; XIC, extracted ion chromatogram

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