Lipid A moiety

Most of the LPS biological activity (pyrogenicity) is associated with its lipid A moiety. This usually consists of six or more fatty acids attached directly to sugars such as glucosamine. Again, as is the case in relation to the carbohydrate component, lipid A moieties of LPS isolated from different bacteria can vary somewhat. The structure of E coli s lipid A has been studied in the greatest detail its exact structure has been elucidated and it can be chemically synthesized. 

Endotoxin. Endotoxin is the lipopolysaccharide that comprises a major portion of the cell wall of the gram-negative bacteria. The endotoxins from each species of bacteria are different but the lipid A moiety is similar for the Enterobacterlaceae, and has a similar series of biological actions regardless of its source. The lipid A material is different in some of the gram-negative rods present in cotton (i.e. in the Pseudomonas species). This variety of compounds makes quantitation of endotoxin difficult. Hence, it is usually measured by its biological activity as compared to a standard endotoxin (usually that of E. coll). (See Table VIII.)... 

Extraction of lipid A from LPS takes advantage of the acido-labile ketosidic bond between the lipid A and the ketodeoxyoctanoate (Kdo) in the LPS. Acid and heat are sufficient to disrupt the linkage. The lipid A is insoluble in water and therefore can be readily collected by centrifugation. Harsh hydrolysis such as 0.1 M hydrochloric acid at 100°C, and milder hydrolysis treatment with 1 % acetic acid have been used to liberate lipid A moiety from LPS molecules (Fensom and Meadow, 1970 Morrison and Leive, 1975 Oertelt et al., 2001 Osborn, 1963). The harsh hydrolytic conditions could result in partially dephosphorylation and O-deacylation of lipid A (Karibian et al., 1995). This could affect the biological activities of the lipid A, but is useful for the extraction of monophosphoryl lipid A (Qureshi et al., 1982). Milder hydrolysis conditions, such as sodium acetate at pH 4.5, have been proved to be efficient to cleave the lipid A-polysaccharide bond (Rosner et al., 1979). When the hydrolysis is ineffective, 1% SDS can be added to the system (Caroff et al., 1988). The lipid A can be extracted from the hydrolytic reaction mixture using the solvent of chloroform and methanol (2 1, v/v). 

The gel filtration chromatography is employed as an important technique to isolate heterogeneous LPS extracted from bacterial cells, especially for isolation of truncated LPS without lipid A moiety. The truncated LPS can be obtained by hydrolysis with 1% acetic acid at 100°C for 1 h, or 0.1 M NaOH in 99% ethanol at 37°C for 45 min (Chester and Meadow, 1975 Muller-Seitz et al., 1968 Prehm et al., 1975). The former releases the polysaccharide from the lipid A moiety with exclusive spitting of the extremely acid-labile Kdo linkage (Luderitz et al., 1966 Muller-Seitz et al., 1968) the latter removes ester-linked fatty acids from the lipid A, reducing non-polar interactions between LPS components and facilitating their separation (Chester and Meadow, 1975). 

DEAE-cellulose ion-exchange chromatography, which is dependent on the phosphate, pyrophosphate or phosphoethanolamine groups of the lipid A moiety, is widely used for lipid A isolation (El Hamidi et al., 2005 Raetz and Kennedy, 1973). TLC, as a lipids detection method, also can be applied for lipid A isolation (Zhou et al., 1999). In addition, the chromatography techniques based on the molecular... 

Fig. 9.1 The structure of lipopolysaccharide, LPS. LPS consists of an O-specific antigen, a core oligosaccharide and the lipid A moiety. The core oligosaccharide, which varies from one bacterial species to another, is made up of outer and inner sugar regions. Lipid A virtually always includes two glucosamine residues modified by phosphates and a variable number of fatty acid chains (Frecer et al., 2000). The LPS structure was kindly contributed by Professor Helmut Brade (Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Medical and Biochemical Mikrobiology Parkallee 22, D-23845 Borstel, Germany)...

Structure and Properties of H. pylori and C. jejuni Lipid A Moieties... 

Our long-term goal has been to utilize the structural information in the LPS-PMB complex to rationally design non-peptide, small-molecule LPS-sequestrants. We have elected to focus on targeting the lipid A moiety of LPS rather than on downstream inflammatory processes which have all met with failure (Quezado et al., 1995 Zeni et al., 1997). We first elucidated the solution structure of PMB, both in its free, aqueous, as well as LPS-bound states (Fig. 12.6) (Bhattacharjya et al., 1997). We initially evaluated peptides, both naturally occurring (Bhattacharjya et al., 1997 David et al., 1992, 1993), and de novo synthesized (Bhattacharjya et al., 1997 David, 2001), testing specific hypotheses pertaining to structural correlates... 

The TLR4-MD2 hetero-dimer has complex ligand specificity. It can be activated by structurally diverse LPS molecules, and minor changes in synthetic derivatives of LPS can abolish their endotoxic potency (Raetz and Whitfield, 2002 Rietschel et al., 1994). The diversity in potency of LPS is derived from variance within lipid A, as observed in both the number and the length of fatty acid side chains and the presence of terminal phosphate residues with a variety of modifications. Optimal lipid A potency is achieved with bi-phosphorylated, hexa-acylated, lipid A species (Raetz and Whitfield, 2002). Lipid A moieties that deviate from this pattern often demonstrate a significant decrease in endotoxic activity (Alexander and Rietschel, 2001). 

Structural analysis of LOS from a mutant of Xcc defective in core completion revealed that this mutant had modifications in the lipid A moiety, which had reduced acylation and was further derivatised with phosphoryl ethanolamine residues (Dow et al., 1995 Silipo et al., 2008). These changes in lipid A structure abolished the ability to trigger innate immune responses in Arabidopsis (Silipo et al., 2008). Importantly these findings indicate that Xcc has the capacity to modify the structure of its lipid A to reduce its activity as a MAMP in plants (Silipo et al., 2008). It is not known whether these (or other) modifications to lipid A occur when bacteria are within plants. 

The modification and activation of LPS for conjugation to a protein is more complex than for CPS. The first step is detoxification by fragmentation of the lipid A moiety either by the cleavage of the acid-sensitive KDO-lipid A linkage or by saponification of fatty acids. Acid hydrolysis completely eliminates lipid A and could be used in the absence of acid-sensitive monosaccharides for isolation of the 0-polysaccharide. Saponification of fatty acids by base hydrolysis or hydrazinolysis also reduces the toxicity to acceptable levels. 

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