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.
Highlights
The LIPID MAPS Consortium is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin
The total lipid extract was fractionated by normal-phase silica chromatography, yielding a mixture of major and minor Kdo2-Lipid A species (Fig. 1)
Subsequent separation of the hexa-acylated material from the other molecular species was achieved by reverse-phase chromatography, followed by DEAE cellulose chromatography to remove t-butyl ammonium phosphate (TBAP) and remaining pigments [22]
Summary
The LIPID MAPS Consortium (www.lipidmaps. org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. We report the large-scale purification, structural analysis, and biological characterization of a chemically defined LPS, consisting of lipid A and an attached 3-deoxy-Dmanno-octulosonic acid (Kdo) disaccharide (Fig. 1, compound A) [6].
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