Submitted on January 13, 2006
Revised on February 7, 2006
Accepted on February 13, 2006
Purification and properties of Escherichia coli Kdo2-lipid A, 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
Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093
Corresponding Author: edennis{at}ucsd.edu
The Lipid Metabolites And Pathways Strategy (LIPID MAPS) Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all known and novel lipids of the macrophage, following activation by endotoxin. The long-term goal is to quantify the 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 preparation of the highest possible purity and analytical specification is crucial. We now report a new, large-scale preparation of the saccharolipid glycan, Kdo2-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to that of native LPS. Kdo2-Lipid A was extracted with chloroform and methanol from ~2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by column chromatography on silica, DEAE-cellulose, and C18 reverse phase resin. Structure and purity were evaluated by electrospray ionization mass spectrometry (ESI/MS), liquid chromatography-mass spectrometry (LC/MS), 1H-NMR, and elemental analysis. Its bioactivity was compared to that of native 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 micro-array based gene expression profiling, were employed as functional readouts. Kdo2-Lipid A is comparable to LPS in potency and spectrum by these criteria. Its activity is reduced by >103 in cells from TLR-4 deficient mice. The advantage of Kdo2-Lipid A over LPS is that it is a reproducible, defined natural product, and it can be detected by ESI/MS at the low concentrations used to stimulate animal cells. The purity of Kdo2-Lipid A should also facilitate the structural analysis of its complexes with signaling receptors, such as TLR-4/MD2.
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