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J. Biol. Chem., Vol. 279, Issue 24, 25420-25429, June 11, 2004
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From the
Department of Biochemistry and the Duke NMR Spectroscopy Center and Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710, the ¶Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the ||Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia, and the **Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, Paris, 75015, France
Leptospira interrogans differs from other spirochetes in that it contains homologs of all the Escherichia coli lpx genes required for the biosynthesis of the lipid A anchor of lipopolysaccharide (LPS). LPS from L. interrogans cells is unusual in that it activates TLR2 rather than TLR4. The structure of L. interrogans lipid A has now been determined by a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR spectroscopy, and biochemical studies. Lipid A was released from LPS of L. interrogans serovar Pomona by 100 °C hydrolysis at pH 4.5 in the presence of SDS. Following purification by anion exchange and thin layer chromatography, the major component was shown to have a molecular weight of 1727. Mild hydrolysis with dilute NaOH reduced this to 1338, consistent with the presence of four N-linked and two O-linked acyl chains. The lipid A molecules of both the virulent and nonvirulent forms of L. interrogans serovar Icterohaemorrhagiae (strain Verdun) were identical to those of L. interrogans Pomona by the above criteria. Given the selectivity of L. interrogans LpxA for 3-hydroxylaurate, we propose that L. interrogans lipid A is acylated with R-3-hydroxylaurate at positions 3 and 3' and with R-3-hydroxypalmitate at positions 2 and 2'. The hydroxyacyl chain composition was validated by gas chromatography and mass spectrometry of fatty acid methyl esters. Intact hexa-acylated lipid A of L. interrogans Pomona was also analyzed by NMR, confirming the presence a 
-1',6-linked disaccharide of 2,3-diamino-2,3-dideoxy-D-glucopyranose units. Two secondary unsaturated acyl chains are attached to the distal residue. The 1-position of the disaccharide is derivatized with an axial phosphate moiety, but the 4'-OH is unsubstituted. 1H and 31P NMR analyses revealed that the 1-phosphate group is methylated. Purified L. interrogans lipid A is inactive against human THP-1 cells but does stimulate tumor necrosis factor production by mouse RAW264.7 cells.
Received for publication, January 20, 2004 , and in revised form, March 1, 2004.
* This work was supported by National Institutes of Health Grants GM-51310 and GM-51796 (to C. R. H. R.) and GM-54882 (to R. J. C.), by Grant PTR94 from the Institut Pasteur (to C. W.), and by a grant from the National Health and Medical Research Council of Australia (to B. A.). The Duke NMR Center is partially supported by National Institutes of Health Grant NCI P30-CA-14236. NMR instrumentation in the Duke NMR Center was funded by the National Science Foundation, the National Institutes of Health, the North Carolina Biotechnology Center, and Duke University. 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: Dept. of Biochemistry, Duke University Medical Center, P.O. Box 3711, Durham, NC 27710. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.
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