Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide

Description of a lipid A containing a hybrid backbone of 2-amino-2-deoxy-D-glucose and 2,3-diamino-2,3-dideoxy-D-glucose

Authors


Correspondence to U. Zähringer, Forschungsinstitut Borstel. Parkallee 22, W-2061 Borstel, Federal Republic of Germany

Abstract

The chemical structure of Campylobacter jejuni CCUG 10936 lipid A was elucidated. The hydrophilic backbone of the lipid A was shown to consist of three (1 → 6)-linked bisphosphorylated hexosamine disaccharides. Neglecting the phosphorylation pattern, a D-glucosamine (2-amino-2-deoxy-D-glucose) disaccharide [β-D-glucosaminyl-(1 → 6)-D-glucosamine], a hybrid disaccharide of 2,3-diamino-2,3-dideoxy-D-glucose and D-glucosamine [2,3-diamino-2,3-dideoxy-β-D-glucopyranosyl-(1 → 6)-D-glucosamine], and a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide were present in a molar ratio of 1:6:1.2. Although the backbones are bisphosphorylated, heterogeneity exists in the substitution of the polar head groups. Phosphorylethanolamine is α-glycosidically bound to the reducing sugar residue of the backbone, though C-1 is also non-stoichiometrically substituted by diphos-phorylethanolamine. Position 4′ of the non-reducing sugar residue carries an ester-bound phosphate group or is non-stoichiometrically substituted by diphosphorylethanolamine. By methylation analysis it was shown that position 6′ is the attachment site for the polysaccharide moiety in lipopolysaccharide. These backbone species carry up to six molecules of ester- and amide-bound fatty acids. Four molecules of (R)-3-hydroxytetradecanoic acid are linked directly to the lipid A backbone (at positions 2, 3, 2′, and 3′). Laser desorption mass spectrometry showed that both (R)-3-hydroxytetradecanoic acids linked to the non-reducing sugar unit carry, at their 3-hydroxyl group, either two molecules of hexadecanoic acid or one molecule of tetradecanoic and one of hexadecanoic acid. It also suggested that the (R)-3-(tetradecanoyloxy)-tetradecanoic acid was attached at position 2′, whereas (R)-3-(hexadecanoyloxy)-tetradecanoic acid was attached at position 3′, or at positions 2′ and 3′. Therefore, the occurrence of three backbone disaccharides differing in amino sugar composition and presence of a hybrid disaccharide differentiate the lipid A of this C. jejuni strain from enterobacterial and other lipids A described previously.

Abbreviations
14:0

tetradecanoic acid

16:0

hexadeanoic acid, 14:(3-OH). (R)-3-hydroxytetradecanoic

14:0(3-OMe)

(R)-3-methoxytetradecanoic acid

 

GlcNAc-ol 2-acetamido-2-deoxy-D-glucitol

GlcN3N

2,3-diamino-2,3-dideoxy-D-glucose

GlcNAc3NAc and GlcNAc3NAc-ol

2,3-di-N-acetamido-2,3-dideoxy-D-glucose and D-glucitol

LPS-HFred

de-O-acylated, dephosphorylated, and reduced lipopolysaccharide

HVPE

high-voltage paper electrophoresis

dOclA

3-deoxy-D-manno-2-octulosonic acid

Neu5Ac

N-acetyl-néuraminic acid

lipid AHCl and lipid NaOAc

free lipid A obtained from lipopolysaccharide by mild acid hydrolysis using 0.1 M HCl or 0.1 M acetate buffer pH 4.4

lipid Ared

reduced lipid A

lipid A-HFred

de-O-acylated, dephosphorylated, and reduced lipid A

CI-MS. EI-MS

GLC-MS and LDMS, chemical-ionization, electron-impact, gas liquid chromatography and laser desorption mass spectrometry

Enzyme
 

Alkaline phosphatase (EC 3.1.3.1)