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J. Biol. Chem., Vol. 278, Issue 36, 34090-34101, September 5, 2003

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Structural Analysis of Oligosaccharides from Lipopolysaccharide (LPS) of Escherichia coli K12 Strain W3100 Reveals a Link between Inner and Outer Core LPS Biosynthesis^*

Sven Müller-Loennies , Buko Lindner and Helmut Brade

From the Borstel Research Center, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115–154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional -D-GlcN in 17 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L--D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an -Kdo-(24)-[-L-Rha-(15)]--Kdo-(24)--Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L--D-Hep-(16)--D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.

Received for publication, April 16, 2003 , and in revised form, June 16, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants DFG LI-448 (to B. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Borstel Research Center, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany. Tel.: 49 4537 188 (ext. 467); Fax.: 49 4537 188 (ext. 419); E-mail: sml{at}fz-borstel.de.

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