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J. Biol. Chem., Vol. 276, Issue 42, 39492-39500, October 19, 2001
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From The Hemophilus influenzae Hap adhesin
is an autotransporter protein that undergoes an autoproteolytic
cleavage event resulting in extracellular release of the adhesin domain
(Haps) from the membrane-associated translocator domain
(Hap
The Hemophilus influenzae Hap Autotransporter Is
a Chymotrypsin Clan Serine Protease and Undergoes
Autoproteolysis via an Intermolecular Mechanism*
§,
,§**
The Edward Mallinckrodt Department of Pediatrics
and § Department of Molecular Microbiology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
¶ Department of Microbiology, University of Texas Southwestern
Medical Center, Dallas, Texas 75235-9048
). Hap autoproteolysis is mediated by
Ser243 and occurs at LN1036-7 and to a lesser extent
at more COOH-terminal alternate sites. In the present study, we sought
to further define the mechanism of Hap autoproteolysis. Site-directed
mutagenesis of residues His98 and Asp140
identified a catalytic triad conserved among a subfamily of
autotransporters and reminiscent of the SA (chymotrypsin) clan of
serine proteases. Amino-terminal amino acid sequencing of
histidine-tagged Hap species and site-directed
mutagenesis established that autoproteolysis occurs at LT1046-7,
FA1077-8, and FS1067-8, revealing a consensus target sequence for
cleavage that consists of ((Q/R)(A/S)X(L/F)) at the
P4 through P1 positions. Examination of a recombinant strain co-expressing a Hap derivative lacking all cleavage sites
(Hap
1036-99) and a Hap derivative lacking proteolytic activity
(HapS243A) demonstrated that autoproteolysis occurs by an
intermolecular mechanism. Kinetic analysis of Hap autoproteolysis in
bacteria expressing Hap under control of an inducible promoter
demonstrated that autoproteolysis increases as the density of Hap
precursor in the outer membrane increases, confirming intermolecular
cleavage and suggesting a novel mechanism for regulation of bacterial
adherence and microcolony formation.
*
This work was supported by an Established Investigator Award
from the American Heart Association (to J. W. S.), a research grant from the March of Dimes (to J. W. S.), United States
Public Health Service Grant AI17621 (to E. J. H.), and funds
from Wyeth-Lederle Vaccines.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.
Current address: Virus and Cell Biology Department, Merck & Co., Inc., West Point, PA 19486.
**
To whom corresponding should be addressed: Dept. of Pediatrics,
Washington University School of Medicine, 660 South Euclid Ave., Campus
Box 8208, St. Louis, MO 63110. Tel.: 314-286-2887; Fax: 314-286-2895;
E-mail: stgeme@borcim.wustl.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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