WO2000063394A2 - Un polypeptide presentant une activite amidolytique vis-a-vis d'une serpine - Google Patents

Un polypeptide presentant une activite amidolytique vis-a-vis d'une serpine Download PDF

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WO2000063394A2
WO2000063394A2 PCT/US2000/010574 US0010574W WO0063394A2 WO 2000063394 A2 WO2000063394 A2 WO 2000063394A2 US 0010574 W US0010574 W US 0010574W WO 0063394 A2 WO0063394 A2 WO 0063394A2
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polypeptide
amino acid
isolated
serpin
seq
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WO2000063394A3 (fr
WO2000063394A9 (fr
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James Travis
Jan Potempa
Daniel Nelson
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University of Georgia Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • 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 E. gingivalis may have a physiological role in modulating the human immune system in addition to their general ability to degrade proteins.
  • arginine-specific gingipain A RgpA
  • arginine-specific gingipain B Rgp B
  • Kgp lysine-specific gingipain
  • 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)).
  • E. gingivalis lipopolysaccharide has been shown to increase mRNA levels of interleukin-8 (IL-8) in neutrophils (Sugita et al., Inflammation. 22(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..
  • E. gingivalis has the ability to inhibit both IL-8 accumulation in gingival epithelial cells (Darveau et al., Infect. Immirn.. 66(4): 1660- 1665 (1998)), as well as transepithelial migration (Madianos et al., Infect. Immun.. 65(10):3983-3990 (1997)).
  • E. 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.
  • 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)).
  • the bacterial outer membrane of E. 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 elastase (HNE) and cathepsin G.
  • HNE human neutrophil elastase
  • these proteinases may cause abnormal connective tissue destruction
  • the presence of human plasma proteinase inhibitors (serpins) typically ⁇ mize connective tissue destruction by complexing with endogenous proteinases. These complexes are ultimately absorbed by the liver for degradation.
  • high protein levels of the ⁇ , -proteinase inhibitor ( ⁇ ,-PI) have been detected in GCF samples from patients diagnosed with severe periodontal disease (Huynh et al., J. Clin.
  • a polypeptide particularly an oral bacterial polypeptide that interacts with a serpin such as the human serpin 0.,-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.
  • the deduced amino acid sequence of periodontain as determined by both partial peptide sequencing of the purified polypeptide and characterization of the E. gingivalis genome is provided.
  • the present invention provides an isolated oral bacterial polypeptide which has amidolytic activity for cleavage of a nondenatured human ⁇ , -proteinase inhibitor at a reactive site loop region of the inhibitor.
  • the isolated polypeptide demonstrates amidolytic activity in a solution containing about 1 mM to about 500 mM Tris, about 500 ⁇ M to about 100 mM cysteine maintained at a pH of about 7 to about 8.
  • 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 S ⁇ Q 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 S ⁇ Q ID NO: 1. More preferably, the isolated polypeptide contains an amino acid sequence having a percentage amino acid identity of greater than 52% to that of amino acid 148 to amino acid 629 of S ⁇ Q ID NO: 1.
  • the isolated polypeptide has an amino acid sequence represented by S ⁇ Q ID NO: 1, an active analog or an active fragment thereof, and more preferably, an amino acid sequence represented by amino acid 148 to amino acid 843 of SEQ ID NO: 1, an active analog or an active fragment thereof.
  • the present invention further provides an isolated nucleic acid encoding an oral bacterial polypeptide which has amidolytic activity for cleavage of a nondenatured human ⁇ , -proteinase inhibitor at a reactive site loop region of the inhibitor. Also provided is an isolated nucleic acid encoding a polypeptide which 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 present invention further provides an isolated nucleic acid encoding a polypeptide which 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 nucleic acid fragment of the invention preferably has a nucleotide sequence represented by SEQ ID NO: 2.
  • the isolated nucleic acid fragment of the invention further preferably encodes a polypeptide having an amino acid sequence with a percentage amino acid identity of greater than 37% when compared to amino acid 148 to amino acid 843 of SEQ ID NO: 1.
  • the present invention also provides an isolated nucleic acid fragment as described herein wherein the complement of the nucleic acid fragment hybridizes to SEQ ID NO: 2 under hybridization conditions of 0.5 M phosphate buffer, pH 7.2, 7% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetra- acetate (EDTA), at 68° C, followed by three 20 minute washes in 2x SSC (IX SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, at 65° C, wherein at least about 15 nucleotides of the complement hybridize.
  • SDS sodium dodecyl sulfate
  • EDTA mM ethylenediaminetetra- acetate
  • the present invention further provides a method for identifying an inhibitor of a polypeptide which has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of a serpin.
  • the method includes isolating an agent that inhibits the amidolytic activity of the polypeptide by incubating the polypeptide with the agent under conditions that promote amidolytic activity of the polypeptide and determining if the amidolytic activity of the polypeptide is reduced relative to the amidolytic activity of the polypeptide in the absence of the agent.
  • the polypeptide is isolated form Porphyromonas gingivalis.
  • the invention also provides an immunogenic composition having a polypeptide which has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of a serpin and is capable of eliciting antibodies in an animal.
  • composition containing an inhibitor to a polypeptide that has amidolytic activity for cleavage of a nondenatured serpin at a reactive site loop region of a serpin isolated from an oral bacterium.
  • Proteinase “Proteinase,” “peptidase,” and “protease” all refer to enzymes that catalyze the hydrolysis of peptide bonds in a polypeptide.
  • a “peptide bond” or “amide bond” is a covalent bond between the alpha-amino group of one amino acid and the alpha-carboxyl group of another amino acid.
  • isolated means that a polypeptide is either removed from its natural environment or synthetically derived.
  • the polypeptide is purified, i.e., essentially free from any other polypeptides and associated cellular products or other impurities.
  • target polypeptide includes any denatured polypeptide and/or a native polypeptide, i.e., nondenatured, that contains at least one exposed contiguous amino acid region, such as a random coil as determined for example, by x-ray crystallography, that is susceptible to cleavage, preferably by periodontain.
  • the exposed amino acid region typically contains at least about 3 contiguous amino acids, and preferably at least about 10 contiguous amino acids.
  • Periodontain target polypeptides include, for example, serpin polypeptides in general, and mammalian serpin polypeptides specifically.
  • an “active analog” or “active fragment” of a polypeptide of the invention is one that has amidolytic activity by hydrolysis of a peptide bond present in the target polypeptide as described herein. Active analogs and active fragments are described in greater detail herein.
  • the aligned putative gene products of prtT (SEQ ID NO: 5) and periodontain (SEQ ID NO: 1), both deduced from P. gingivalis strain W83 genome, and streptopain (SEQ ID NO: 6), from S. pyogenes, were aligned according to multiple sequence alignment using the sequence alignment program CLUSTAL W and the percent amino acid identity was determined by BLAST 2 SEQUENCES at NCBI using the default matrix.
  • the black box(es) indicate that the same amino acid is present in a compared sequence as indicated, and grey box(es) indicate that a similar amino acid, e.g., a conservative amino acid substitution, is present in a compared sequence as indicated.
  • Cell associated activities (clear bar), vesicle associated activities (hatched bar), and soluble activities (solid bar) were normalized to 100% for each strain.
  • periodontain and other polypeptides of the invention are additionally characterized by the nonspecific proteolysis of a target polypeptide in that a specific amino acid residue residing in the polypeptide does not necessarily dictate the specificity of the proteinase.
  • periodontain will cleave any target polypeptide nonspecifically in a denaturing environment.
  • a "denaturing environment,” as used herein, includes in vitro environment conditions, for example, heat, strong reducing agents, such as dithiothreitol and ⁇ -mercaptoethanol, sodium dodecyl sulfate, or other chemical manipulation including carboxymethylate-maleylate treatment.
  • the present invention is directed to a nucleic acid fragment encoding a polypeptide, particularly a cysteine proteinase, active analog (including active modification) or active fragment thereof.
  • the nucleic acid fragment can have a nucleotide sequence as shown in SEQ ID NO: 2.
  • the percentage nucleotide identity is the higher of the following two numbers: (a) the number of nucleotides that the two sequences have in common within the alignment, divided by the number of nucleotides in SEQ ID NO: 2, multiplied by 100; or (b) the number of nucleotides that the two sequences have in common within the alignment, divided by the number of nucleotides in the candidate coding region, multiplied by 100.
  • two nucleic acid sequences have at least about 50%, more preferably at least about 70%, and most preferably at least about 90% identity.
  • the coding region can then be isolated and ligated into a vector as described below.
  • An expression vector can optionally include a Shine Dalgarno site (e.g., a ribosome binding site), and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the enzyme. It can also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis.
  • the nucleic acid fragment used to transform the host cell can optionally further include a transcription termination sequence.
  • the rrnB terminators which is a stretch of DNA that contains two terminators, TI and T2, is an often used terminator that is incorporated into bacterial expression systems (J. Brosius et al., J. Mol. Biol.. 148:107-127 (1981)).
  • the immunogenic composition can further include excipients or diluents that are pharmaceutically acceptable as carriers and compatible with the immunogenic composition.
  • pharmaceutically acceptable carrier refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the immunogenic composition may contain minor amovmts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and or adjuvants which enhance the effectiveness of the immune-stimulating composition.
  • the immunogenic composition can be used in a method for protecting an animal from a disease caused by E. gingivalis.
  • This method includes administering the immunogenic composition and eliciting antibodies to a cysteine proteinase, antigenic analog, antigenic fragment, or antigenic modification.
  • the diseases that can be treated in this manner include periodontal diseases, which includes gingivitis and periodontitis. Clinical hallmarks of periodontitis include lose of tooth attachment and periodontal pocket formation.
  • the present invention also includes a method for identifying and isolating an inhibitor of a polypeptide of the invention.
  • the method includes identifying an agent that inhibits the amidolytic activity of the polypeptide by incubating the polypeptide with the agent under conditions that promote amidolytic activity of the polypeptide and determining if the amidolytic activity of the polypeptide is reduced relative to the amidolytic activity of the polypeptide in the absence of the agent.
  • Example 1 Isolation and characterization of a proteinase derived from P. gingivalis
  • native ⁇ , -PI (0.15 nanomoles (nmols)
  • samples containing a putative inactivating activity in assay buffer 50 mM Tris, 10 mM cysteine, pH 7.8 and allowed to incubate at time intervals of 0, 30, 60, 90, 120, 240 minutes after which an equimolar amount of ⁇ -chymotrypsin (0.15 nmols) was added to complex any remaining functional 0.,-PI.
  • Endpoint absorbance at 405 nm was read in a Molecular Devices SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA) and the percentage of ⁇ ,-PI remaining was calculated from controls (no 0C ⁇ -PI, 0.15 nM ⁇ - chymotrypsin) versus (0.15 nM 0.,-PI, 0.15 nM ⁇ -chymotrypsin) in the 4 minute reaction with N-Suc-Ala-Ala-Pro-Phe-/>flr ⁇ -nitroanalide.
  • ⁇ ,-PI Inactivation of ⁇ ,-PI by periodontain was further determined by electrophoresis. Briefly, ⁇ ,-PI (2 ⁇ M) was incubated with periodontain (2 nM) for 0, 15, 30, 45, and 60 minutes before the reaction was stopped by the addition of an SDS sample buffer. A control sample was preincubated with 100 ⁇ M of L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane (E-64) for 5 minutes prior to 60 minutes incubation with 0.,-PI. The samples were electrophoresed on a 12% separating gel. Subsequently, samples were separated on a 16% peptide gel to isolate a 3 kD fragment produced by this cleavage.
  • both N-te ⁇ ninal amino acid sequence analysis and mass spectroscopy of the isolated fragment revealed that the major cleavage site was after the glutamic acid residue and a minor cleavage site was after the phenylalanine relative to the Met onine-Serine bond which forms the "bait" region of the reactive site loop (RSL) and is attacked by HNE at the (*) position.
  • RSL reactive site loop
  • the dialyzed fraction was clarified by centrifugation (40,000 x g, for 30 minutes), concentrated by ultrafiltration (Amicon PM-10 membrane, Millipore, Bedford, MA) and applied in 20 ml fractions, each representing 5 L of starting supernatant, to a Sephadex G-150 column (5 x 105 centimeters (cm)) (Amersham-Pharmacia, Uppsala, Sweden) equilibrated with buffer A, at a flow rate of 30 milliliter/hour (ml/h).
  • the activity was pooled, dialyzed against 50 mM Tris, pH 7.4 (buffer B), and further purified by ion exchange chromatography on a Mono-Q column (FPLC system, Amersham-Pharmacia, Uppsala, Sweden), with elution in a linear gradient of 0 mM to 500 mM NaCl in buffer B.
  • Activity was concentrated and final purification obtained by separation on a TSK-GEL G3000S W (TosoHaas, Montgomeryville, PA) column using 50 mM Tris, 200 mM NaCl, pH 7.4.
  • PVDF polyvinylidene difluoride
  • periodontain was used at a concentration which was capable of completely inactivating 0.15 nmols of ⁇ ,-PI in our standardized assay (Nelson et al., Anal. Biochem.. 260:230-236 (1998)) after one hour of incubation.
  • the cleavage of inhibitor (%) was normalized to a native inhibitor control in order to give the relative percent inactivation for each compound or protein tested against periodontain.
  • inhibitor (20 ⁇ g) was incubated with 1 ⁇ g of periodontain for 4 hours, after which the sample was subjected to 16% SDS-PAGE to separate the approximately 3 kD fragment obtained by cleavage within the loop.
  • the fragment was analyzed for both amino terminal sequence and molecular mass using an automated protein sequencer and mass spectroscopy, respectively, as described above.
  • the gel was washed twice with 2.5 % weight volume (w/v) Triton X-100 to remove the SDS and then incubated in activation buffer (50 mM Tris, 20 mM cysteine, pH 7.4) at 37 °C for two hours.
  • activation buffer 50 mM Tris, 20 mM cysteine, pH 7.4
  • the zymograph was developed in 0.1% Amido Black, with clearing zones (negative staining) indicating proteolytic digestion of the incorporated gelatin.
  • the gelatin zymograph indicated that the periodontain heavy chain (55 kD) contained the catalytic active site.
  • the DNA fragment coding for the N-te ⁇ ninus of periodontain was amplified by PCR using Pwo DNA Polymerase (Boehringer-Mannheim/Hof&nan LaRoche Ltd., Basel, Switzerland), and 10 nanograms (ng) of W50 P. gingivalis DNA (purified by the Purgene kit, Gentra Systems Inc., Madison, MN). PCR was carried out with 500 ng of primers for 1 minute at 94 °C, 1 minute at 65 °C, and 20 seconds at 72 °C.
  • the expected product of 69 basepairs was purified from a 2% agarose gel with the Ultrafree MC Millipore Filter (Millipore, Bedford, MA), phosphorylated at the 5' end with polynucleotide kinase (PNK), and blunt end ligated into a Smal digested pUC19 vector.
  • the fragment coding for the N- teraiinus of periodontain was identified by sequence analysis.
  • the gene encoding periodontain was identified using the NCBI open reading frame (ORF) finder program (also found at NCBI) and the amino acid sequence, obtained by conceptual translation of the entire ORF, was further used for homology screening performed with the NCBI BLAST search tool.
  • ORF open reading frame
  • Periodontain activity was detected over a broad pH range of about 6.0 to about 9.0, with the optimum being between a pH of about 7.5 to about 8.0.
  • the enzyme was stable at 37 °C, overnight, and at 4 °C for several weeks, when stored in the absence of cysteine.
  • the presence of reducing agent resulted in a 50% loss of activity at 37 °C overnight, presumably because of autodigestion. Heating to 60 °C caused complete loss of activity.
  • Samples were routinely stored at -80 °C for several months with less than a 10% loss in activity.
  • Periodontain was completely inactive in the absence of reducing agents, whereas full activity was achieved with either free cysteine, ⁇ -mercaptoethanol, dithiothreitol (DTT), or dithioerythritol (DTE) at 0.1 mM concentration. Unlike the gingipains, which have higher activity in free cysteine, no single reducing agent was superior in activating periodontain. Furthermore, increasing concentrations of these reagents (up to 10 mM) did not cause any additional stimulation of periodontain activity.
  • periodontain Based on its requirement for a reducing environment to become active, periodontain can be classified as a cysteine proteinase, and this is confirmed by the fact that it is readily inhibited by common cysteine proteinase inhibitors (Table II). The ability of E-64 to inhibit periodontain suggests that this enzyme is more closely related to members of the papain family than other cysteine proteinases of P. gingivalis which are either not inhibited (Kgp) or only weakly inhibited (Rgp's) by this compound. Table II. Inhibition Profile of Periodontain
  • the region encoding the N-terminal sequence of the 55 kD catalytic subunit of periodontain was amplified by PCR using degenerate primers and P. gingivalis W50 DNA.
  • the 69 basepair PCR product was cloned and sequenced.
  • the gene encoding periodontain was extracted from the unfinished microbial genome at NCBI and found to encode an 843 amino acid residue protein with a calculated molecular weight of 93,127 Da ( Figure 1).
  • the predicted size of the translated protein is approximately 20 kD larger than that found experimentally by both gel filtration and SDS-PAGE.
  • the native protein would have a predicted molecular weight of 76,727 Da, composed of a 52,981 Da catalytic heavy chain and a 23,764 Da C- terminal light chain, with a calculated pi of 5.18.
  • This is in agreement with the experimental findings of a native protein of about 70 to about 80 kD by gel filtration, composed of a catalytic subunit of 55 kD and a noncatalytic subunit of 23 kD by SDS-PAGE, with a pi of 5.3.
  • These data suggest a potential role in the processing of the pro-form of periodontain by both gingipains R and gingipain K, all of which are abundantly present in P gingivalis.
  • the regulating inhibitor of HN ⁇ is 0.,-PI, a plasma protein which forms a complex with this proteinase and is rapidly removed from the circulation and degraded (Mast et al., J. Biol. Chem.. 266(24):15810-15816 (1991)).
  • the inhibitor can itself be inactivated by either oxidation at its reactive site or by proteolytic cleavage by nontarget proteinases within the RSL region
  • Periodontain was purified from E. gingivalis culture fluids and was found to be a cysteine proteinase that is apparently produced as a heterodimer.
  • This enzyme is similar to the other characterized cysteine proteinases from E. gingivalis in that all are primarily secreted in strain HG66, yet present on membranes and vesicles in all other strains.
  • Each has a signal peptide sequence, a long prepropeptide, a large catalytic domain, approximately 50 kD, and an additional C-te ⁇ ninal extension (about 20 kD to about 40 kD).
  • periodontain is characterized by an apparently nonspecific proteolysis of peptides or denatured proteins.
  • ⁇ ,-PI a native protein
  • Periodontain and prtT containing 843 and 840 amino acid residues, respectively, are more than twice the size of the streptopain (398 residues) ( Figure 1)
  • the function of the light chain of periodontain is presently unknown; however, the C-terminal domain for the putative prtT gene product has recently been shown to be identical to the putative product for a hemin regulated gene, hemR (Karunakaran et al., J. BacterioL. 179(6):1898-1908 (1997)).
  • gingipains which only have a specificity for either Lys-X or Arg-X bonds, would be restricted in their ability to degrade large proteins to the size of dipeptides or tripeptides which could then be transported into the bacterium. Therefore, it is possible that a number of broadly specific proteinases and proteinase, including periodontain, may be physiologically important to E. gingivalis, not necessarily as virulence factors, but rather for nutrient acquisition. This may be accomplished by three pathways. First, since periodontain is the only peptidase in E.
  • gingivalis so far described which possesses the ability to inactivate ⁇ ,-PI, this maybe a mechanism for increasing the levels of HN ⁇ , a nonspecific host proteinase which might be utilized in protein degradation.
  • periodontain may augment the degradation of peptides produced by the actions of the gingipains.
  • E. gingivalis produces a prolyl dipeptidyl peptidase (Kiyama et al., Bioch. Bioph. Acta. 1396:39-46 (1998)), a prolyl tripeptidyl peptidase, and an uncharacterized collagenase. This proteolytic milieu in the GCF could aid in the final production of peptides capable of being taken up by E. gingivalis.

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Abstract

La présente invention concerne un polypeptide bactérien oral isolé présentant une activité amidolytique de clivage de polypeptides dénaturés et des polypeptides de serpine non dénaturés et notamment un polypeptide inhibiteur de la protéinase-α1 humaine. Le polypeptide mûr de l'invention présente un poids moléculaire d'environ 70 kD à environ 80 kD. L'invention concerne également une séquence d'acides nucléiques codant le polypeptide bactérien oral de l'invention, et des procédés d'identification d'inhibiteurs du polypeptide et des compositions du type compositions immunogènes et compositions inhibitrices.
PCT/US2000/010574 1999-04-21 2000-04-20 Un polypeptide presentant une activite amidolytique vis-a-vis d'une serpine Ceased WO2000063394A2 (fr)

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US10/030,330 US6833262B1 (en) 1999-04-21 2000-04-20 Polypeptide having amidolytic activity for a serpin
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WO2005042770A3 (fr) * 2003-11-03 2005-07-07 Ethicon Inc Methodes, peptides, et biocapteurs utiles pour detecter un large spectre de bacteries

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JP2001526035A (ja) * 1997-12-10 2001-12-18 シーエスエル、リミテッド Ｐｏｒｐｈｏｒｙｍｏｎａｓｇｉｎｇｉｖａｌｉｓポリペプチドおよびヌクレオチド

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005042770A3 (fr) * 2003-11-03 2005-07-07 Ethicon Inc Methodes, peptides, et biocapteurs utiles pour detecter un large spectre de bacteries
JP2007514409A (ja) * 2003-11-03 2007-06-07 エチコン・インコーポレーテツド 細菌の広域スペクトルの検出に有用な方法、ペプチドおよびバイオセンサー
JP4864715B2 (ja) * 2003-11-03 2012-02-01 エチコン・インコーポレーテツド 細菌の広域スペクトルの検出に有用な方法、ペプチドおよびバイオセンサー
US8609358B2 (en) 2003-11-03 2013-12-17 Systagenix Wound Management (Us), Inc. Methods, peptides and biosensors useful for detecting a broad spectrum of bacteria
US9315851B2 (en) 2003-11-03 2016-04-19 Woundchek Laboratories (Us), Inc. Methods, peptides, and biosensors useful for detecting a broad spectrum of bacteria

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