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标题：Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4
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作者：Raetz, Christian R. H. ; Garrett, Teresa A. ; Reynolds, C. Michael 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2006
卷号：47
期号：5
页码：1097-1111
DOI：10.1194/jlr.M600027-JLR200
出版社：American Society for Biochemistry and Molecular Biology
摘要：The LIPID MAPS Consortium (www.lipidmaps.org) is developingcomprehensive procedures for identifying all lipids of the macrophage,following activation by endotoxin. The goal is to quantify temporaland spatial changes in lipids that occur with cellular metabolismand to develop bioinformatic approaches that establish dynamiclipid networks. To achieve these aims, an endotoxin of the highestpossible analytical specification is crucial. We now reporta 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-LipidA was extracted from 2 kg cell paste of a heptose-deficientEscherichia coli mutant. It was purified by chromatography onsilica, DEAE-cellulose, and C18 reverse-phase resin. Structureand purity were evaluated by electrospray ionization/mass spectrometry,liquid chromatography/mass spectrometry and ¹H-NMR. Its bioactivitywas compared with LPS in RAW 264.7 cells and bone marrow macrophagesfrom wild-type and toll-like receptor 4 (TLR-4)-deficient mice.Cytokine and eicosanoid production, in conjunction with geneexpression profiling, were employed as readouts. Kdo₂-LipidA is comparable to LPS by these criteria. Its activity is reducedby >10³ in cells from TLR-4-deficient mice. The purity ofKdo₂-Lipid A should facilitate structural analysis of complexeswith 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 ${alpha}$ , tumor necrosis factor- ${alpha}$ ; XIC, extracted ion chromatogram