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J. Biol. Chem., Vol. 281, Issue 38, 27765-27772, September 22, 2006

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Cloning of a Cholesterol--glucosyltransferase from Helicobacter pylori^*

Anne-Helene Lebrun¹, Christian Wunder¹², Janosch Hildebrand¹, Yuri Churin, Ulrich Zähringer^¶, Buko Lindner^¶, Thomas F. Meyer, Ernst Heinz, and Dirk Warnecke³

From the Biocenter Klein Flottbek and Botanical Garden, University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany, the Department of Molecular Biology, Max Planck Institute for Infection Biology, Schumannstrasse 21-22, 10117 Berlin, Germany, and the ^¶Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol--glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003–1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl--glucoside, cholesteryl-6'-O-acyl--glucoside, cholesteryl-6'-O-phosphatidyl--glucoside, and cholesteryl-6'-O-lysophosphatidyl--glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol--glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol--glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl--glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl--glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol--glucosyltransferase.

Received for publication, April 7, 2006 , and in revised form, June 29, 2006.

^* This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 470. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contribute equally to this work.

² Present address: Cell Biology and Metabolism Branch, National Institutes of Health, 18 Library Dr., Bethesda, MD 20892.

³ To whom correspondence should be addressed: Biocenter Klein Flottbek and Botanical Garden, University of Hamburg, Ohnhorststr. 18, 22609 Hamburg, Germany. Tel.: 49-40-42816-343; Fax: 49-40-42816-254; E-mail: warnecke{at}botanik.uni-hamburg.de.

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