TY - JOUR
T1 - NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus
AU - Zähringer, Ulrich
AU - Ittig, Simon
AU - Lindner, Buko
AU - Moll, Hermann
AU - Schombel, Ursula
AU - Gisch, Nicolas
AU - Cornelis, Guy R.
N1 - © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2014
Y1 - 2014
N2 - We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-snglycero-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-dimensionalNMRtechniques 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-Dmanno-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. Basedonthese findings, the strict view that lipidAalone represents the toxic center of LPS needs to be reassessed. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
AB - We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-snglycero-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-dimensionalNMRtechniques 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-Dmanno-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. Basedonthese findings, the strict view that lipidAalone represents the toxic center of LPS needs to be reassessed. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
UR - http://www.scopus.com/inward/record.url?scp=84906536740&partnerID=8YFLogxK
U2 - 10.1074/jbc.M114.571489
DO - 10.1074/jbc.M114.571489
M3 - Article
C2 - 24993825
SN - 0021-9258
VL - 289
SP - 23963
EP - 23976
JO - The Journal of Biological Chemistry
JF - The Journal of Biological Chemistry
IS - 34
ER -