O-deacylation of lipid

Kawasaki, K., Ernst, R.K., Miller, S.I. 3-O-deacylation of lipid A by PagL, a PhoP/PhoQ-regulated deacylase of Salmonella typhimurium, modulates signaling through Toll-like receptor 4. J Biol Chem 279 (2004) 20044-20048. 

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). 

Trent, M., Pabich, W., Raetz, C., Miller, S. A PhoP/PhoQ-induced Lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium. J Biol Chem 276 (2001a) 9083-9092. 

Hydroxylaminolysis, treatment with stronger alkali (0.5 m NaOH, 2 h, 100°C) and alkaline methanolysis (0.25 m NaOMe, 1 h, 50°C) lead to complete O-deacylation of LPS and lipid A (176). Particularly in the case of alkaline methanolysis, ester-linked 3-acyloxyacyl residues undergo, in addition to transmethylation, a -elimination reaction, whereby the (R)-3-hy-droxy fatty acid ester is first transformed into the a,/ -unsaturated and then into the (S.-R -methoxy fatty acid methyl ester. The acyl substituent, on the other hand, is eliminated in the form of the free fatty acid (176). In fact, the presence of a 3-methoxyacyl derivative in the fatty acid spectrum of a given LPS is a strong indication for the presence of an ester-bound 3-acyloxyacyl... 

Some bacteria, such as Haemophilus influenzae and N. meningitidis, express R-type LPS comprising a heterogeneous mixture of core oligosaccharides. LPS can be subjected either to O-deacylation to remove O-linked fatty acids from lipid A or to mild acid treatment to release the entire lipid A. These treatments increase the solubility for MS analysis and simplify the MS data for the elucidation of the structure of molecules. 

ES-MS and ES-MS-MS (negative ion mode) were used by Gibson et al. (1996) for the structural determination of LOSs. Both O-deacylated and intact LOS were analysed. Mass spectra from O-deacylated LOS were more readily interpretable than mass spectra obtained from intact LOS. The paper is focused on the heterogeneity of the oligosaccharide moiety rather than on the fatty acid composition of the lipid A region. 

HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program allows to identify the positions of O-acylation process. The new approach was successfully implemented by Widmalm et al. in the structural elucidation of the O-antigen PS of E. coli 059. The algorithm used in this methodology was demonstrated as flowchart in Fig. 2a and the top-ranked structure of repeating unit of O-deacetylated repeating unit of the O-antigen PS of E. coli 059 in Fig. 2b. 

For the analysis of fatty acids amide-linked to GlcN(I), several chemical degradation procedures are available. One comprises periodate oxidation of 0-deacylated and 2H-reducedlipid A (lipid A-OH ). Following permethyl-ation, g.l.c.-m.s. analysis revealed a 2-deoxy-1 -deuterio-1,3-di-O-methyl-2-(iV-methyl-3-methoxyacylamido)glycerol derivative in which the amide-finked fatty acid at GlcN(I) was identified as 14 0(3-OH) in B. pertussis (93), and as 16 0(3-OH) and 18 0(3-OH) in R. trifolii 81). 

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