Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.
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.
Department of Biochemistry, Duke University Medical Center, Durham, NC, USA.
Christian R H
CR
Raetz
Teresa A
TA
Garrett
C Michael
CM
Reynolds
Walter A
WA
Shaw
Jeff D
JD
Moore
Dale C
DC
Smith
Anthony A
AA
Ribeiro
Robert C
RC
Murphy
Richard J
RJ
Ulevitch
Colleen
C
Fearns
Donna
D
Reichart
Christopher K
CK
Glass
Chris
C
Benner
Shankar
S
Subramaniam
Richard
R
Harkewicz
Rebecca C
RC
Bowers-Gentry
Matthew W
MW
Buczynski
Jennifer A
JA
Cooper
Raymond A
RA
Deems
Edward A
EA
Dennis
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D008070:Lipopolysaccharides / Q000302:isolation & purification / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects*; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D015230:Prostaglandin D2 / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D051197:Toll-Like Receptor 4 / Q000502:physiology*
C506188:Kdo2-lipid A; D008070:Lipopolysaccharides; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D015230:Prostaglandin D2