Purification and characterization of a collagen-degrading protease from Porphyromonas gingivalis.
A trypsin-like protease was purified from spent culture medium of oral pathogen Porphyromonas gingivalis by chromatography on columns of DEAE-Sepharose, gel filtration on Sephadex G-100, and chromatofocusing on PBE-94. Purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 55,000. Purified protease hydrolyzed type I, III, IV, and V collagen from human placenta, and type I collagen from rat tail and calf skin, but did not hydrolyze type II collagen from chicken sternal cartilage. The purified enzyme also hydrolyzed the C3 component of complement, fibrinogen, fibronectin, alpha 1-antitrypsin, alpha 2-macroglobulin, apotransferrin, and human serum albumin. The hydrolytic activity of the purified enzyme on chromogenic substrates was limited to substrates with arginine in the P-1 position, although synthetic peptides were also cleaved at Lys-X linkage. The enzyme was activated by reducing agents dithiothreitol, L-cysteine, and glutathione and inhibited by cysteine protease inhibitors N-ethylmaleimide, iodoacetic acid, and iodoacetamide. The enzyme was also inhibited by trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), leupeptin, antipain, salivary histidine-rich protein (HRP-5), soybean trypsin inhibitor, and EDTA. Since the protease is able to degrade the connective tissue components of periodontal tissue as well as components of host defense mechanism, this enzyme may be a potent virulence factor of P. gingivalis involved in invasion and tissue destruction.
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Magainin Pharmaceuticals Inc., Plymouth Meeting, Pennsylvania 19462.
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D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
D005353:Fibronectins; D011480:Protease Inhibitors; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; D005340:Fibrinogen; D003094:Collagen; D010450:Endopeptidases; D014357:Trypsin