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J. Biol. Chem., Vol. 278, Issue 14, 12101-12108, April 4, 2003
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From the Departments of Helicobacter
pylori secretes a toxin, VacA, that can form anion-selective
membrane channels. Within a unique amino-terminal hydrophobic region of
VacA, there are three tandem GXXXG motifs (defined by
glycines at positions 14, 18, 22, and 26), which are characteristic of
transmembrane dimerization sequences. The goals of the current study
were to investigate whether these GXXXG motifs are required
for membrane channel formation and cytotoxicity and to clarify the role
of membrane channel formation in the biological activity of VacA. Six
different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A,
and G26A) were introduced into the unique hydrophobic region located
near the amino terminus of VacA. The effects of these mutations were
first analyzed using the TOXCAT system, which permits the study of
transmembrane oligomerization of proteins in a natural membrane
environment. None of the mutations altered the capacity of
ToxR-VacA-maltose-binding protein fusion proteins to insert into a
membrane, but G14A and G18A mutations markedly diminished the capacity
of the fusion proteins to oligomerize. We then introduced the six
alanine substitution mutations into the vacA chromosomal
gene of H. pylori and analyzed the properties of purified
mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced
vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A
did not. Subsequent experiments examined the capacity of each mutant
toxin to form membrane channels. In a planar lipid bilayer assay, VacA
proteins containing G13A, G22A, and G26A mutations formed
anion-selective membrane channels, whereas VacA proteins containing
P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A,
and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A,
VacA-G14A, and VacA-G18A did not. These data indicate that an intact
proline residue and an intact
G14XXXG18 motif within the
amino-terminal hydrophobic region of VacA are essential for membrane
channel formation, and they also provide strong evidence that membrane
channel formation is essential for VacA cytotoxicity.
Essential Role of a GXXXG Motif for Membrane
Channel Formation by Helicobacter pylori Vacuolating
Toxin*
,,¶
**
Medicine and
¶ Microbiology and Immunology, Vanderbilt University School of
Medicine and the Veterans Affairs Medical Center, Nashville,
Tennessee 37232, and the § Department of Molecular
Physiology and Biological Physics and Biophysics Program, University of
Virginia School of Medicine, Charlottesville, Virginia
22908
*
This work was supported in part by National Institutes of
Health Grants AI39657 and DK53623 and by the Medical Research
Department of the Department of Veterans Affairs.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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