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J Biol Chem, Vol. 275, Issue 5, 3713-3721, February 4, 2000
From the Department of Microbiology and Immunology, Uniformed
Services University of the Health Sciences,
Bethesda, Maryland 20814
Shiga toxin variant type 2d (Stx2d) produced by
some strains of Shiga toxin-producing Escherichia coli is
composed of an enzymatically active A subunit and a B (binding)
pentamer. The cytotoxicity of Stx2d is increased (activated)
10-1000-fold for Vero cells when the toxin is incubated with mucus
obtained from the small intestine of mice. In this study we isolated an
Stx2d activator and identified it as a mouse elastase with strong
homology to human elastase IIIB. Moreover, commercially available
porcine pancreatic elastase preparations also activated Stx2d
cytotoxicity although with a lower specific activity than isolated
mouse elastase. Elastase directly nicked the Stx2d A subunit to
A1 and A2, an event that did not correlate with
activation. However, elastase also reduced the size and changed the
isoelectric point of the A2 peptide, as determined by
SDS-polyacrylamide gel electrophoresis and two-dimensional
electrophoresis followed by Western immunoblot analysis. This
elastase-mediated size and charge shift in the A2 peptide
of Stx2d occurred concurrently with activation of the toxin. Both the
reduction in size of the Stx2d A2 peptide by incubation with elastase as well as the associated activation of Stx2d
cytotoxicity were fully inhibited by elastatinal, an
elastase-specific inhibitor.
Elastase in Intestinal Mucus Enhances the Cytotoxicity of Shiga
Toxin Type 2d*
,
*
This work is supported by Public Health Service Grant
AI20148-16 from the National Institutes of Health and Uniformed
Services University of the Health Sciences Protocol R073EQ. The W. M.
Keck Biomedical Mass Spectrometry Laboratory is funded by a grant from the W. M. Keck Foundation, and the University of Virginia Biomedical Research Facility is funded by a grant from the University of Virginia
Pratt Committee.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.
Present address: Biosynexus Inc., Rockville, MD 20850.
§
To whom correspondence should be addressed: Dept. of Microbiology
and Immunology, Uniformed Services University of the Health Sciences,
4301 Jones Bride Rd., Bethesda, MD 20814. Tel.: 301-295-3400; Fax:
301-295-3773; E-mail: aobrien@usuhs.mil.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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