Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-01-25
2001-08-14
Slobodyansky, Elizabeth (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S220000
Reexamination Certificate
active
06274718
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention is bacterial proteases, more particularly those of
Porphyromonas gingivalis,
most particularly the arginine-specific proteases termed Arg-gingipains herein and the nucleotide sequences encoding same.
BACKGROUND OF THE INVENTION
Porphyromonas gingivalis
(formerly
Bacteroides gingivalis
) is an obligately anaerobic bacterium which is implicated in periodontal disease.
P. gingivalis
produces proteolytic enzymes in relatively large quantities; these proteinases are recognized as important virulence factors (Smalley et al. (1989)
Oral Microbiol. Immun.
4, 178-179; Marsh et al. (1989)
FEMS Microbiol, Lett.
59, 181-186; Grenier and Mayrand (1987)
J. Clin. Microbiol,
25, 738-740]. A number of physiologically significant proteins, including collagen [Birkedal-Hansen et al. (1988)
J. Periodontal Res.
23, 258-264; Sundqvist et al. (1987)
J. Periodontal Res.
22, 300-306]; fibronectin [Wikstrom and Linde (1986)
Infect. Immun.
51, 707-711; Uitto et al. (1989)
Infect. Immun.
57, 213-218]; immunoglobulins [Kilian, M. (1981)
Infect. Immun.
34, 757-765; Sundqvist et al. (1985)
J. Med. Microbiol,
19, 85-94; Sato et al. (1987)
Arch. Oral Biol.
32, 235-238]; complement factors C3, C4, C5, and B [Sundqvist, et al. 1985) supra; Schenkein, H. A. (1988)
J. Periodontal Res.
23, 187-192); lysozyme (Otsuka et al. (1987)
J. Periodontal Res.
22, 491-498); iron-binding proteins (Carlsson et al. (1984)
J. Med. Microbiol,
18, 39-46); plasma proteinase inhibitors (Carlsson et al. (1984)
Infect. Immun.
43, 644-648; Herrmann et al. (1985)
Scand. J. Dent. Res.
93, 153-157); fibrin and fibrinogen (Wikstrom et al. (1983)
J. Clin. Microbiol.
17, 759-767; Lantz et al. (1986)
Infect. Immun.
54, 654-658); and key factors of the plasma coagulation cascade system (Nilsson et al. (1985)
Infect. Immun.
50, 467-471), are hydrolyzed by proteinases from this microorganism. Such broad proteolytic activity may play a major role in the evasion of host defense mechanisms and the destruction of gingival connective tissue associated with progressive periodontitis (Saglie et al. (1988)
J. Periodontol.
59, 259-265).
Progressive periodontitis is characterized by acute tissue degradation promoted by collagen digestion and a vigorous inflammatory response characterized by excessive neutrophil infiltration (White and Maynard (1981)
J. Periodontal Res.
16, 259-265). Gingival crevicular fluid accumulates in periodontitis as gingival tissue erosion progresses at the foci of the infection, and numerous plasma proteins are exposed to proteinases expressed by the bacteria at the injury site. It was speculated that neutrophils may have been recruited to the gingiva, in part, by the humoral chemotactic factor C5a. The complement components C3 and C5 are activated by complex plasma proteases with “trypsin-like” specificities called convertases (Muller-Eberhard (1988)
Ann. Rev. Biochem.
57, 321-347). The human plasma convertases cleave the &agr;-chains of C3 and C5 at a specific site generating biologically active factors known as anaphylatoxins (i.e. C3a and C5a). The anaphylatoxins are potent proinflammatory factors exhibiting chemotactic and/or spasmogenic activities as well as promoting increased vascular permeability. The larger products from C3 and C5 cleavage (i.e. C3b and C5b) participate in functions including complement cascade activation, opsinization, and lytic complex formation.
There are conflicting data as to the number and types of proteinases produced by
P. gingivalis.
In the past, proteolytic activities of
P. gingivalis
were classified into two groups; those enzymes which specifically degraded collagen and the general “trypsin-like” proteinases which appeared to be responsible for other proteolytic activity. Trypsin (and trypsin-like proteases) cleaves after arginine or lysine in the substrates (See, e.g. Lehninger A. L. (1982),
Principles of Biochemistry,
Worth Publishing, Inc., New York). The Arg-gingipain described herein differ in that they are specific for cleavage after only arginine, with no activity for cleavage after lysine residues.
More recently, Birkedal-Hansen et al. (Birkedal-Hansen et al. (1988) supra.) performed a systematic analysis of the effect of six classes of proteinase inhibitors on Porphyromonas collagenolytic activity which strongly suggested that all proteinases from this organism are dependent on free cysteine groups and metal ions, as indicated by inhibition by thiol-blocking reagents and metal chelators. On the other hand, Grenier et al. (Grenier et al. (1989)
Infect Immun.
57, 95-99) identified at least eight proteolytic enzymes with molecular masses in the range of 29-110 kDa. Two of these appeared to be serine proteinases with glycyl-prolyl peptidase activity, one of which appears to be about 29 kDa (Grenier and McBride (1987)
Infect. Immun.
55, 3131-3136).
All other enzymes were shown to be activated by cysteine and hydrolyzed the synthetic substrate Benzoyl-L-Arginyl-p-Nitroanilide (Bz-L-Arg-p-NA). Whether this represent distinct proteolytic enzymes or autocatalytic products of a single proteinase remains to be established. Although many attempts have been made to separate one of these trypsin-like proteinases (Otsuka, et al. (1987) supra.; Ono et al. (1987)
Oral Microbiol. Immunol.
2, 77-81; Fujimura and Nakamura (1987)
Infect. Immun.
55, 716-720; Suido et al. (1987)
J. Periodontal Res.
22, 412-418; Tsutsui et al. (1987)
Infect. Immun.
55, 420-427; Uitto, V. J. (1987)
J. Periodontal Res.
22, 58-63; Sorsa et al. (1987)
J. Periodontal Res.
22, 375-380) until now none has been purified sufficiently for rigorous biochemical and enzymological characterization.
Several communications have reported the cloning of other protease genes from
P. gingivalis.
(Arnott et al. (1990)
Arch. Oral Biol.
35: 97S-99S; Bourgeau et al. (1992)
Infect. Immun.
60: 3186-3192; Kato et al. (1992)
J. Bacteriol.
174: 3889-3895; Otogoto et al. (1993)
Infect. Immun.
61: 117-123; Park et al. (1992)
FEMS Microbiol. Lett.
92: 273-278; Pratt et al. (1981)
Nucleic Acids Res.
9: 4459-4474; Takahashi et al. (1991)
FEMS Microbiol. Lett.
84: 135-138).
In this application, 50 kDa and a high molecular weight trypsin-like, thiol-activated proteinases of
P. gingivalis,
which have been purified to apparent homogeneity for the first time, is described and termed Arg-gingipain herein, and the nucleotide sequence of the gene encoding it is provided.
There is a need in the art for purified Arg-gingipain, for example, as antigen for preparing antibodies specific to this protein or for vaccines useful in protection against periodontal disease, and for studies to identify inhibitors of this enzyme.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a proteinase preparation comprising a substantially pure low molecular weight Arg-gingipain, termed Arg-gingipain-1 (or gingipain-1), herein, said gingipain-1 having an apparent molecular mass of 50 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent molecular mass of 44 kDa as estimated by gel filtration chromatography, said gingipain-1 having amidolytic and proteolytic activity for cleavage after arginine residues and having no amidolytic and/or proteolytic activity for cleavage after lysine residues, wherein the amidolytic and/or proteolytic activity is inhibited by cysteine protease group-specific inhibitors including iodoacetamide, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain,trans-epoxysuccinyl-L-leucylamido-(4-guanidine) butane, TLCK, TPCK, p-aminobenzamidine, N-chlorosuccinamide, and chelating agents including EDTA and EGTA, wherein the amidolytic and/or proteolytic activity of said gingipain-1 is not sensitive to inhibition by human cystatin C, &agr;2-macroglobulin, &agr;1-proteinase inhibitor, antithrombin III, &agr;2-antiplasmin, serine protease group-specific inhibitors including diisopropylfluorophosphate, phenylmethyl sulfonylfluoride and 3
Barr Philip J.
Pavloff Nadine
Potempa Jan Stanislaw
Travis James
Greenlee Winner and Sullivan PC
Slobodyansky Elizabeth
University of Georgia Research Foundation Inc.
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