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

被引:190
作者
Raetz, Christian R. H.
Garrett, Teresa A.
Reynolds, C. Michael
Shaw, Walter A.
Moore, Jeff D.
Smith, Dale C., Jr.
Ribeiro, Anthony A.
Murphy, Robert C.
Ulevitch, Richard J.
Fearns, Colleen
Reichart, Donna
Glass, Christopher K.
Benner, Chris
Subramaniam, Shankar
Harkewicz, Richard
Bowers-Gentry, Rebecca C.
Buczynski, Matthew W.
Cooper, Jennifer A.
Deems, Raymond A.
Dennis, Edward A.
机构
[1] Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92093 USA
[2] Univ Calif San Diego, Dept Cellular & Mol Med, La Jolla, CA 92093 USA
[3] Univ Calif San Diego, Dept Pharmacol, La Jolla, CA 92093 USA
[4] Duke Univ, Med Ctr, Dept Biochem, Durham, NC USA
[5] Avanti Polar Lipids Inc, Alabaster, AL USA
[6] Univ Colorado, Dept Pharmacol, Hlth Sci Ctr, Aurora, CO USA
[7] Scripps Res Inst, Dept Immunol, La Jolla, CA 92093 USA
[8] Univ Calif San Diego, Dept Bioengn, La Jolla, CA 92093 USA
关键词
3-deoxy-D-manno-octulosonic acid-Lipid A; Escherichia coli; Re; endotoxin; macrophage; mass spectrometry; LIPID MAPS; toll-like receptor-4;
D O I
10.1194/jlr.M600027-JLR200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-octulo-sonic 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 H-1-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(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.
引用
收藏
页码:1097 / 1111
页数:15
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