title: NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus.
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.
authors.affiliation: From the Division of Immunochemistry/Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany, uzaehr@fz-borstel.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zähringer
authors.affiliation: Infection Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland, and simon.ittig@unibas.ch.
authors.forename: Simon
authors.initials: S
authors.lastname: Ittig
authors.affiliation: From the Division of Immunochemistry/Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany.
authors.forename: Buko
authors.initials: B
authors.lastname: Lindner
authors.affiliation: From the Division of Immunochemistry/Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany.
authors.forename: Hermann
authors.initials: H
authors.lastname: Moll
authors.affiliation: From the Division of Immunochemistry/Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Schombel
authors.affiliation: From the Division of Immunochemistry/Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gisch
authors.affiliation: Infection Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland, and the Department of Biology, University of Namur, B5000 Namur, Belgium.
authors.forename: Guy R
authors.initials: GR
authors.lastname: Cornelis
mesh_terms: D002206:Capnocytophaga / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data
chemical_list: D008070:Lipopolysaccharides
keywords: Capnocytophaga canimorsus; DHPC Micelles; Glycolipid Structure; Gram-negative Bacteria; High-performance Liquid Chromatography (HPLC); Lipopolysaccharide (LPS); Nuclear Magnetic Resonance (NMR)