Cloning of a Cholesterol-α-glucosyltransferase from Helicobacter pylori

Anne-Helene Lebrun,Ernst Heinz,Christian Wunder,Yuri Churin,Janosch Hildebrand,Dirk Warnecke,Thomas F. Meyer,Ulrich Zähringer,Buko Lindner

doi:10.1074/jbc.m603345200

Abstract

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-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-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-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-alpha-glucosyltransferase.

Highlights

Pathogenic bacteria, which do not produce cholesterol, but take it up from their hosts, are able to glycosylate cholesterol by innate glycosyltransferases (6 –12)
To identify the enzyme, which is responsible for the biosynthesis of cholesteryl-␣-glucoside in H. pylori, we have employed a strategy based upon both insertional inactivation in H. pylori and heterologous expression in E. coli and P. pastoris
This has led to the characterization of HP0421, a glycosyltransferase of the glycosyltransferase family 4 (GT4) family [35], the members of which display a retaining chemistry

Summary

NDb ND ND ND ppm

5.2c lished for the 5␣,8␣-epidioxyergosta-6,22-dienyl-␤-D-glucopyranoside [39]. From these data we conclude that expression of HP0421 in P. pastoris results in the biosynthesis of ergosta5,7,22-trienyl-3-␣-D-glucopyranoside (ergosteryl-␣-glucoside, ␣-EG) and 5␣,8␣-epidioxyergosta-6,22-dienyl-3-␣-D-glucopyranoside (epidioxyergosteryl-␣-glucoside). A parallel expression of a bacterial GT4 member from T. maritima in P. pastoris led to the biosynthesis of diacylglycerol-␣glucoside as described before [34] These data demonstrate that HP0421 from H. pylori is a sterol-␣-glucosyltransferase. E. coli cells expressing HP0421 were disrupted by ultrasonication and the sterol glycosyltransferase activity in cell-free homogenates was measured using radiolabeled substrates. B, cholesterol ␣-glucosyltransferase activity was detected in the cell-free homogenate and in the membrane fraction of H. pylori, but not in the cytosolic fraction. Formly dissolved, but were present as both monomers and components of lipid vesicles, the exact determination of their concentrations in the assay was not possible These results indicate which substrates are accepted or discriminated by the cholesterol-␣-glu-.

Steryl glucoside synthesis

Findings

DISCUSSION