University of Georgia Research Foundation Inc

Emory University

Research, Incorporated

Miles Inc.

Morehouse School of Medicine

James Travis

Jan Stanislaw Potempa

Philip J Barr

Nadine Pavloff

Jan Pohl

William M Shafer

Ralph Pannell

Robert Neil Pike

Agnieszka Banbula

Neelesh Bangalore

Eugen Schnabel

Daniel Craig Nelson

A Christopher Skjold

Murray S Blum

Marcin Bugno

Caroline Attardo Genco

Takahisa Imamura

S Troy Whitworth

J Stanislaw Potempa

The present invention provides isolated polypeptides, prolyl tripeptidyl-peptidases, and active analogs, active fragments or active modifications thereof, having amidolytic activity for cleavage of a peptide bond present in a target peptide having at least 30 amino acids. Isolated nucleic acid fragments encoding isolated prolyl tripeptidyl-peptidases are also provided, as are methods of reducing growth of a bacterium by inhibiting a prolyl tripeptidyl-peptidase.

Prolyl tripeptidyl peptidases nucleic acid of Porphyromonas gingivalis

The present invention provides isolated polypeptides, dipeptidylpeptidases, active analogs, active fragments, or active modifications thereof, having amidolytic activity for cleavage of a peptide bond between the second and third amino acids from the N-terminal end of a target polypeptide, wherein the target polypeptide has an aliphatic or an aromatic residue as a substituent on the &#x3b1;-carbon atom of the second amino acid from the N-terminal end of the peptide. Isolated nucleic acids encoding dipeptidylpeptidases are also provided, as are methods of reducing growth of a bacterium by inhibiting a dipeptidylpeptidase.

Dipeptidylpeptidases and methods of use

An isolated oral bacterial polypeptide having amidolytic activity for cleavage of denatured polypeptides and nondenatured serpin polypeptides and particularly a human &agr; -proteinase inhibitor polypeptide is provided. The mature polypeptide of the invention has a molecular weight of about 70 kD to about 80 kD. Also provided is an isolated nucleic acid sequence encoding the oral bacterial polypeptide of the invention, methods for identifying inhibitors of the polypeptide and compositions such as immunogenic compositions and inhibitor compositions.

Polypeptide having amidolytic activity for a serpin

Methods for controlling blood coagulation, and suitable pharmaceutical compositions that include a polypeptide that enhances the anticoagulation process (or inhibitors thereof for reversing the anticoagulation process) are provided.

Methods and compositions for control of blood coagulation

The present invention provides polypeptides, including chymotrypsin-like polypeptides and elastase-like polypeptides, having amidolytic acitivity for cleavage of a peptide bond present in a target polypeptide. The polypeptides can be isolated from ants, including larvae. Isolated nucleic acid fragments encoding isolated polypeptides are also provided, as are methods of developing and using inhibitors of chymotrypsin-like polypeptides and elastase-like polypeptides.

Ant proteases and methods of inhibition

Provided herein is a nucleotide sequence encoding an Arg-specific gingipain, said Arg-gingipain characterized as 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-guanidino)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 proteolytic activities of ginigipan-1 are stabilized by Ca,and 3,4-diisochlorocoumarin, and wherein the amidolytic and/or wherein the amidolytic and/or proteolytic activities of said gingipain-1 are stimulated by glycine-containing peptides and glycine analogues, and methods for preparation. As specifically exemplified, high and low molecular weight forms of Arg-gingipains and sequences encoding them are prepared from,Said high molecular weight Arg-gingipain comprises a proteolytic component as described hereinabove and at least one hemagglutinin component.

Porphyromonas gingivalis arginine-specific proteinase coding sequences

Provided herein are methods and immunogenic compositions useful for protecting mammals from infection and pathology of P. gingivalis. Specifically, arginine-specific proteases of Porphyromonas gingivalis and peptides derived therefrom offer protection against infection. Immunogenic compositions comprising a 50 kDa arginine-specific protease, the high molecular weight complex or peptides from one of the foregoing proteins are capable of protecting against P. gingivalis infection and/or gingivitis and/or periodontitis caused thereby in mammals, including humans.

Immunogenic compositions comprising porphyromonas gingivalis proteins

Provided herein is a Porphyromonas gingivalis high molecular weight arginine-specific proteinase comprising a protease component of 50 kD and a hemagglutinin component of about 44 kD as estimated by SDS-PAGE. The proteinase is stimulated by glycine containing peptides and glycine analogues. It is inhibited by cysteine protease group specific inhibitors.

Porphyromonas gingivalis arginine-specific proteinase

Peptides which exhibit antimicrobial activity comparable to certain known antibiotics are provided. These peptides are related in sequence to amino acid sequences within Cathepsin G. A broad spectrum bactericidal peptide disclosed herein is RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22). It is active against Pseudomonas aeruginosa, Neisseria gonorrhoeae and Staphylococcus aureus. RRENTQQHITARRAIRHPQY (SEQ ID NO:19) and GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:26) also exhibited potent activity against P. aeruginosa strains, including clinical isolates. IIGGR (SEQ ID NO:1) and IVGGR (SEQ ID NO:2) act against both gram-negative and gram-positive bacterial strains. HPQYNQR (SEQ ID NO:3) and certain related peptides are also active against both gram-negative and gram-positive bacteria, including, but not limited to, strains of Escherichia coli, Neisseria gonorrhoeae, Staphylococcus aureus, Capnocytophage sputigena and Pseudomonas aeruginosa. The peptides of the present invention will be useful in pharmaceutical compositions useful in the treatment of prophylaxis of infections.

Antimicrobial peptides

Provide herein is a substantially pure Lys-gingipain complex preparation, Lys-gingipain being characterized as having an apparent molecular mass of 105 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, where sample is prepared without boiling, said Lys-gingipain having amidolytic and proteolytic activity for cleavage after lysine residues and having no amidolytic and/or proteolytic activity for cleavage after arginine residues, wherein the amidolytic and/or proteolytic activity is inhibited by TLCK, cysteine protease group-specific inhibitors including iodoacetamide and iodoacetic acid, wherein the amidolytic and/or proteolytic activity of said Lys-gingipain is not sensitive to inhibition by leupeptin, antipain, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, serine protease group-specific inhibitors including diisopropylfluorophosphate and phenylmethyl sulfonylfluoride, and antibodies specific for the Lys-gingipain protein complex and its catalytic component, methods for preparation. As specifically exemplified, a Lys-gingipain protein complex is purified from Porphyromonas gingivalis H66, and the 60 kDa catalytic component of the Lys-gingipain protein complex has an amino acid sequence as given in SEQ ID NO:14 from amino acid 1 through amino acid 509. Also provided are nucleic acid sequences encoding this catalytic protein. The nucleotide coding sequence of the 60 kDa catalytic component of the Lys-gingipain protein complex is given in SEQ ID NO:13, from nucleotide 1336 through nucleotide 2863. The Lys-gingipain complex also comprises a hemagglutinin component identified by an N-terminal amino acid sequence as given in SEQ ID NO:14, amino acids 510-714.

Lysine-specific Porphyromonas gingivalis proteinase

Provide herein is a substantially pure gingipain-1 preparation, gingipain-1 being characterized as 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-guanidino)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, .alpha.2-macroglobulin, .alpha.1-proteinase inhibitor, antithrombin III, .alpha.2-antiplasmin, serine protease group-specific inhibitors including diisopropylfluorophosphate, phenylmethyl sulfonylfluoride and 3,4-diisochlorocoumarin, and wherein the amidolytic and/or proteolytic activities of gingipain-1 are stabilized by Ca.sup.2+ and wherein the amidolytic and/or proteolytic activities of said gingipain-1 are stimulated by glycine-containing peptides and glycine analogues, and methods for preparation. As specifically exemplified, Arg-gingipain-1 and Arg-gingipain-2 are purified from Porphyromonas gingivalis, and the mature Arg-gingipain-2 has an amino acid sequence as given in SEQ ID NO:5 from amino acid 1 through amino acid 510. Also provided are nucleic acid sequences encoding Arg-gingipain-2. The nucleotide coding sequence of the mature Arg-gingipain-2 is given in SEQ ID NO:4, from nucleotide 1630 through nucleotide 3105.

Provide herein is a substantially pure Lys-gingipain complex preparation, Lys-gingipain being characterized as having an apparent molecular mass of 105 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, where sample is prepared without boiling, said Lys-gingipain having amidolytic and proteolytic activity for cleavage after lysine residues and having no amidolytic and/or proteolytic activity for cleavage after arginine residues, wherein the amidolytic and/or proteolytic activity is inhibited by TLCK, cysteine protease group-specific inhibitors including iodoacetamide and iodoacetic acid, wherein the amidolytic and/or proteolytic activity of said Lys-gingipain is not sensitive to inhibition by leupeptin, antipain, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, serine protease group-specific inhibitors including diisopropylfluorophosphate and phenylmethyl sulfonylfluoride, and antibodies specific for the Lys-gingipain protein complex and its catalytic component, methods for preparation. As specifically exemplified, a Lys-gingipain protein complex is purified from Porphyromonas gingivalis H66, and the 60 kDa catalytic component of the Lys-gingipain protein complex has an amino acid sequence as given in SEQ ID NO: 14 from amino acid 1 through amino acid 509. Also provided are nucleic acid sequences encoding this catalytic protein. The nucleotide coding sequence of the 60 kDa catalytic component of the Lys-gingipain protein complex is given in SEQ ID NO:13, from nucleotide 1336 through nucleotide 2863. The Lys-gingipain complex also comprises a hemagglutinin component identified by an N-terminal amino acid sequence as given in SEQ ID NO:14, amino acids 510-714.

Peptides which exhibit antimicrobial activity comparable to certain known antibiotics are provided. These peptides are related in sequence to amino acid sequences within Cathepsin G. A broad spectrum bactericidal peptide disclosed herein is RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22). It is active against Pseudomonas aeruginosa, Neisseria gonorrhoeae and Staphylococcus aureus. RRENTQQHITARRAIRHPQY (SEQ ID NO:19) and GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:20) also exhibited potent activity against P, aeruginosa strains, including clinical isolates. IIGGR (SEQ ID NO:1) and IVGGR (SEQ ID NO:2) act against both gram-negative and gram-positive bacterial strains. HPQYNQR (SEQ ID NO:3) and certain related peptides are also active against both gram-negative and gram-positive bacteria, including, but not limited to, strains of Escherichia coli, Neisseria gonorrhoeae, Staphylococcus aureus, Capnocytophage sputigena and Pseudomonas aeruginosa. The peptides of the present invention will be useful in pharmaceutical compositions useful in the treatment of prophylaxis of infections.

Agent for the detection of esterolytic and/or proteolytic enzymes, containing (a) an amino acid ester or peptide ester of a phenol, as the chromogenic enzyme substrate, and (b) an alcohol as the substance which accelerates the enzymatic cleavage of the amino acid ester bond or peptide ester bond of component (a), characterized in that it contains a salt as the accelerating substance.

Analytical process and agents for the detection of esterolytic and/or

A new method of determining oxidized .alpha.-1-proteinase inhibitor in serum or plasma for use in studying the development of chronic obstructive lung disease is disclosed. Levels of oxidized .alpha.-1-proteinase inhibitor indicate the potential for emphysema development with higher levels showing a decrease in lung protection against elastolytic enzymes such as elastase. This method can be used for patients with a potential for chronic obstructive lung disease rather than having to use bronchial lavage methods for such patients. No other method is known to exist for determining oxidized .alpha.-1-proteinase inhibitor in serum or plasma.

Growing community of inventors

Athens, GA, United States of America

James Travis