Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-10-19
2004-12-21
Achutamurthy, Ponnathapura (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S023000, C435S320100, C536S023200
Reexamination Certificate
active
06833262
ABSTRACT:
BACKGROUND
The anaerobe
Porphyromonas gingivalis
(
P. gingivalis
) has been implicated as a major causative organism of adult onset periodontal disease. Enzymes from this organism have been found to degrade several proteins, including, for example, collagen, fibrinogen, immunoglobulins, complement proteins, and fibronectin Recent evidence has shown that three proteinases released from
P. gingivalis
may have a physiological role in modulating the human immune system in addition to their general ability to degrade proteins. These three proteinases are referred to as gingipains and include arginine-specific gingipain A (RgpA) and arginine-specific gingipain B (Rgp B), which are capable of specifically cleaving after arginine residues, and lysine-specific gingipain (Kgp) which is able to specifically cleave after lysine residues. Working in concert, these proteinases have been shown to produce bradykinin from high molecular weight kininogen, either directly or indirectly (kallikrein activation), resulting in the enhancement of vascular permeability (Imamura, et al.,
Infect, Immun.,
63(5):1999-2003 (1995)). This mechanism, which is used to provide nutritional components for the growth and proliferation of
P. gingivalis
, is presumed to be responsible for both the increased gingival crevicular fluid (GCF) and edema clinically noted in periodontal pockets of patients with advanced periodontitis (Darany et al.,
J. Periodontol.,
63:743-747 (1992)).
The interaction of
P. gingivalis
with a host's immune system response has been paradoxical, in that
P. gingivalis
has demonstrated both pro-inflammatory and anti-inflammatory responses. For example,
P. gingivalis
lipopolysaccharide has been shown to increase mRNA levels of interleukin-8 (IL-8) in neutrophils (Sugita et al.,
Inflammation,
2(3):253-267(1998)), and gingipains R have been shown to increase neutrophil chemotaxis by release of C5a from C5 of the complement system (Wingrove et al.,
J. Biol. Chem.,
7(26):18902-18907 (1992)). However, these proteinases are also capable of cleaving the C5a receptor from infiltrating neutrophils (Jagels et al.,
Infect. Immun.,
64(6):198-1991 (1996)), thereby effectively neutralizing localized chemotactic activity. Additionally,
P. gingivalis
has the ability to inhibit both IL-8 accumulation in gingival epithelial cells (Darveau et al.,
Infect. Immun.,
66(4):1660-1665 (1998)), as well as transepithelial migration (Madianos et al.,
Infect, Immun.,
65(10):3983-3990 (1997)).
These apparent activity contradictions may potentially be explained by the compartmentalization of the oral cavity, wherein distal activation of chemotactic components and proximal paralysis of these factors creates a “leukocyte wall” between the periodontal plaque and gingival epithelium (Miyasaki,
J. Periodontol.,
2:761-774 (1991)). Indeed, it has been reported that _soluble gingipains can stimulate IL-8 activity, whereas membrane bound gingipains, with a limited ability to diffuse beyond the plaque surface, completely degrade IL-8 (Mikolajczyk-Pawlinska et al.,
FEBS Lett.,
440:282-286 (1998)).
The recruitment of neutrophils to the “leukocyte wall” through both the increased leakage of blood vessels and a chemotactic gradient at first appear to be suicidal to
P. gingivalis
. However, such a scenario is not likely, as
P. gingivalis
is known to have evolved mechanisms to survive in the presence of neutrophils. For example,
P. gingivalis
proteinases have been shown to degrade C3 complement and immunoglobulins (Schenkein et al.,
J. Immunol.,
154:5331-5337 (1995)), thereby averting opsonization and subsequent detection by a host. Furthermore, gingipain R has been shown to have an inhibitory effect on the oxidative burst utilized by neutrophils to kill microorganisms (Kadowaki et al.,
J. Biol. Chem.,
269(33):21371-21378 (1994)). Similarly, the bacterial outer membrane of
P. gingivalis
may function as an antioxidant sink due to the incorporation of large amounts of heme (Smalley et al.,
J. Biochem.,
331:681-685 (1998)).
Activated neutrophils in the leukocyte wall typically undergo degranulation due to the inability to phagocytize foreign organisms, thereby expelling large quantities of the proteinases human neutrophil elasase (HNE) and cathepsin G. Although these proteinases may cause abnormal connective tissue destruction, the presence of human plasma proteinase inhibitors (serpins) typically minimize connective tissue destruction by complexing with endogenous proteinases. These complexes are ultimately absorbed by the liver for degradation. For example, high protein levels of the &agr;
1
-proteinase inhibitor (&agr;1-PI) have been detected in GCF samples from patients diagnosed with severe periodontal disease (Huynh et al.,
J. Clin. Periodontol.,
19:187-192 (1992)). However, despite the presence of &agr;1-PI a high HNE activity is observed indicating that the &agr;1-PI must be present in either complexed, oxidized, or proteolytically inactivated forms (Uitto et al.,
J. Clin. Periodontol.,
2:30-37 (1996)). This observation is supported by evidence showing that less than 35% of available &agr;1-PI in the GCF is active as an inhibitor (Smith et al.,
Archs. Oral Biol.,
22(4):301-306 (1994)). Additionally, it has been shown that patients with &agr;
1
-PI deficiencies have a significantly higher frequency of periodontal pocket depths ≧5 mm, thereby being predisposed to manifestations of periodontal disease (Fokkems et al.,
J. Clin. Periodontol.,
25:617-623 (1998)). Thus, there is a need for further understanding the interaction of such serpins with other oral bacterial proteinases.
SUMMARY OF THE INVENTION
Described herein are the isolation, purification and characterization of a polypeptide, particularly an oral bacterial polypeptide that interacts with a serpin such as the human serpin &agr;
1
-PI. This polypeptide is referred to as “periodontain,” not only because of its function as a proteinase, but also because this polypeptide may function as a putative factor in the dysregulation of serpin function in the periodontal cavity of an animal. Additionally, the deduced amino acid sequence of periodontain, as determined by both partial peptide sequencing of the purified polypeptide and characterization of the
P. gingivalis
genome is provided.
Accordingly, the present invention provides an isolated oral bacterial polypeptide which has amidolytic activity for cleavage of a nondenatured human (&agr;
1
-proteinase inhibitor at a reactive site loop region of the inhibitor. The isolated polypeptide demonstrates amidolytic activity in a solution containing about 1 nM to about 500 mM Tris, about 500 &mgr;M to about 100 mM cysteine maintained at a pH of about 7 to about 8. Preferably, the polypeptide is isolated from
Porphyromonas gingivalis
and is a cysteine proteinase.
The polypeptide of the invention preferably has a molecular weight of about 70 kD to about 80 kD as determined by gel filtration. The polypeptide of the invention will preferably cleave the reactive site loop region of the inhibitor represented by SEQ ID NO: 4 between glutamine and alanine and also between phenylalanine and leucine.
The present invention also provides an isolated polypeptide that is an oral bacterial cysteine proteinase and has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of the serpin. The isolated polypeptide is preferably isolated from
Porphyromonas gingivalis
. The isolated polypeptide of the invention further has the capability to cleave a target polypeptide nonspecifically.
The present invention further provides an isolated polypeptide that is isolated from
Porphyromonas gingivalis
and has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of the serpin.
The isolated polypeptide preferably contains an amino acid sequence having a percentage amino acid identity of greater than 37% to that of amino acid 148 to amino acid 843 of SEQ ID NO: 1. More preferably, the isolated polypeptide contains an amino acid sequence havi
Nelson Daniel C.
Potempa Jan S.
Travis James
Achutamurthy Ponnathapura
Moore William W.
Mueting Raasch & Gebhardt, P.A.
University of Georgia Research Foundation Inc.
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