NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

Ulrich Zähringer,Simon Ittig,Buko Lindner,Hermann Moll,Ursula Schombel,Nicolas Gisch,Guy R Cornelis

doi:10.1074/jbc.m114.571489

Abstract

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.

Highlights

Rough-type LPS of C. canimorsus is biologically active, whereas lipid A is not
The Core Oligosaccharide in the Y1C12 Mutant Is Representative of Rough-type C. canimorsus 5 WT LPS—The LPS isolated from the Y1C12 mutant was chosen for the structural analysis of the core oligosaccharide for reasons given below
The Y1C12 mutant gave better yield and higher purity for the core oligosaccharide preparation; the structural analysis described here has been performed with compounds isolated from the mutant, which was regarded to be representative of C. canimorsus LPS

Summary

Introduction

Rough-type LPS of C. canimorsus is biologically active, whereas lipid A is not. Results: Purified C. canimorsus rough-type LPS could be analyzed in intact form by NMR. The intact LPS was analyzed by homonuclear (1H) and heteronuclear (1H,13C, and 1H,31P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry It consists of a penta-acylated lipid A with an ␣-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4؅-phosphate. The intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed

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Conclusion