Affiliation:
1. Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana 46202
Abstract
ABSTRACT
PrtP is a major cysteine proteinase of
Porphyromonas gingivalis
. The gene encoding this proteinase,
prtP
, was cloned into the
Escherichia coli-Bacteroides
shuttle vectors pFD288 and pFD340 and was expressed in
Bacteroides
cells, apparently under the control of its own promoter, when in pFD288, or a
Bacteroides
promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several
Bacteroides
species harboring the recombinant plasmids. The proteinase was recovered from
Bacteroides fragilis
ATCC 25285(pFD340-
prtP
) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by
Nα-p
-tosyl-
l
-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-
d
-Tyr-
l
-Pro-
l
-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither
B. fragilis
ATCC 25285(pFD340-
prtP
) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore,
P. gingivalis
W12, ATCC 33277, KDP110, and HG66 with knockout mutations in
prtP
were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other
P. gingivalis
proteins and describes the properties of
P. gingivalis
cells defective in the production of PrtP.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
12 articles.
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