NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus |
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Authors: | Ulrich Z?hringer Simon Ittig Buko Lindner Hermann Moll Ursula Schombel Nicolas Gisch Guy R Cornelis |
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Institution: | From the ‡Division of Immunochemistry/Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 4a, 23845 Borstel, Germany, ;§Infection Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland, and ;the ¶Department of Biology, University of Namur, B5000 Namur, Belgium |
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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, 13C,15N labeling could be avoided. 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 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 ManI 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. |
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Keywords: | Glycolipid Structure Gram-negative Bacteria High-performance Liquid Chromatography (HPLC) Lipopolysaccharide (LPS) Nuclear Magnetic Resonance (NMR) Capnocytophaga canimorsus DHPC Micelles |
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