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

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 . The LIPID MAPS consortium is developing quantitative methods for evaluating the composition, biosynthesis, and function of all macrophage lipids (1). These amphipathic substances not only are structural components of biological membranes but also play important roles in the pathophysiology of inflammation, atherosclerosis, and growth control. Additional lipid functions should emerge from the comprehensive analysis of macrophage lipids. Electrospray ionization/mass spectrometry (ESI/MS) (2, 3), coupled with prefractionation methods like reversephase liquid chromatography (LC), is being applied systematically to set the stage for the seamless integration of lipid metabolism into the broader fields of genomics, proteomics, and systems biology. To facilitate this endeavor, LIPID MAPS has introduced a new comprehensive classification system for biological lipids, amenable to computer-based data processing and substructure comparison (4). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids,

Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide

“…The fragmentation pattern of the unmodified Kdo 2 -lipid A from cells grown with ambient air (Fig. 4C) displayed the expected fragment ions (22), corresponding to the lipid A fragment (½M-H − at m∕z 1,796.211), the Kdolipid A fragment (½M-2H 2− at m∕z 1,007.632), the Kdo 2 fragment (½M-H 2 O-H − at m∕z 439.109), and the Kdo fragment (½M-H − at m∕z 237.062 and its dehydrated form ½M-H 2 O-H − at m∕z 219.052). With the putative Ko-Kdo-lipid A product generated in the presence of 16 O 2 , the peak attributed to ½M-H − of lipid A was seen at m∕z 1,796.209, the same as with Kdo 2 -lipid A (Fig.…”

“…3A), which were used to calibrate the spectrum (22). In the constructs expressing YpKdoO or BakdoO, a doubly-charged species was seen at m∕z 1125.664 or 1125.658, respectively ( Fig.…”

“…1A, left structure). This substance can be extracted from cells with chloroform/methanol mixtures along with the glycerophospholipids and analyzed by thin layer chromatography (TLC) (22), as shown for the vector control WBB06/pWSK29 (Fig. 2).…”

Structural basis for the acyl chain selectivity and mechanism of UDP- N -acetylglucosamine acyltransferase

“…1) of LPS is required for the growth of most Gram-negative bacteria and accounts for the some of the toxic side-effects of Gram-negative sepsis (1)(2)(3)(4). In Escherichia coli, UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis (5-7), the reversible transfer of the R-3-hydroxymyristoyl moiety from R-3-hydroxymyristoyl-acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc ( Fig.…”

The costimulatory immunogen LPS induces the B-Cell clones that infiltrate transplanted human kidneys

“…To more precisely determine the chemical composition of the minimal epitope that the antibodies reacted with, we took advantage of an elegant series of E. coli mutants studied by the late Christian R. Raetz and his colleagues (32,33). Essentially, the biosynthetic pathway of LPS requires sequential activation of various α-glycosyltranferases and mutations in these enzymes leads to accumulation of truncated core oligosaccharides of defined structure (Fig.…”