Characterization of lipid A from Pseudomonas aeruginosa O-antigenic B band lipopolysaccharide by 1D and 2D NMR and mass spectral analysis

D. Nedra Karunaratne, James C. Richards, Robert Hancock

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Abstract

The Lipid A from the lipopolysaccharide of Pseudomonas aeruginosa was examined by high-field nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The backbone structure and the position of phosphate substituents were unambiguously established by one- and two-dimensional 1H, 13C, and 31P NMR techniques on a de-O-acylated Lipid A sample. The Lipid A has a β-(1,6)-glucosamine disaccharide structure which is substituted by phosphomonoesters through glycosidic bonds at C-1 and at C-4′. The configuration of the glycosidically linked phosphate at position C-1 was identified as α which is the same as that of Enterobacterial Lipid A. Chemical analysis revealed that the Lipid A contained 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, 3-hydroxydecanoic, and hexadecanoic acids in the approximate molar ratios 2.2:2.0:0.2:0.8:0.4. From 1H NMR and fast atom bombardment (FAB) mass spectrometry on the de-O-acylated Lipid A, it was established that both glucosamine residues were N-acylated by 3-hydroxydodecanoic acid. The identity and positions of the ester bound fatty acids in the intact Lipid A were investigated by negative ion FAB-MS. In addition to the hexaacyl and pentaacyl Lipid A species, a tetraacyl species was identified. Heterogeneity due to hydroxylated and nonhydroxylated dodecanoic acid esters could be uniquely localized to the nonreducing β-glucosamine residue from the fragmentation pattern observed in the negative ion FAB-MS. The complete structure of the Lipid A from P. aeruginosa will be useful in understanding the determinants responsible for the endotoxin activity of this molecule.

Original languageEnglish
Pages (from-to)368-376
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume299
Issue number2
DOIs
Publication statusPublished - 1 Jan 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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