Raetz CR, Garrett TA, Reynolds CM, 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 Kdo2-lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4 Journal of Lipid Research47(5) (2006)
1097-1111
NCBI PubMed ID:16479018 Journal NLM ID:0376606 Publisher: ASBMB Institutions: Department of Biochemistry, Duke University Medical Center, PO Box 3711, Durham, NC, Avanti Polar Lipids, Inc., Alabaster, AL, USA, 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, Department of Bioengineering, Department of Chemistry and Biochemistry and Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
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
Escherichia coli, endotoxin, mass spectrometry, Re, macrophage, 3-deoxy-D-manno-octulosonic acid-Lipid A, LIPID MAPS, toll-like receptor-4
Structure type: oligomer Location inside paper: p.1099,fig.1 Aglycon: lipid A Trivial name: Kdo2-lipid A Contained glycoepitopes:IEDB_130650,IEDB_130659
Methods: NMR, MS Biological activity: biological activity data
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