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Purification and characterization of a collagen-degrading protease from Porphyromonas gingivalis.

1994; Elsevier BV; Volume: 269; Issue: 1 Linguagem: Inglês

10.1016/s0021-9258(17)42391-x

1083-351X

Gurrinder S. Bedi, Tom A. Williams,

Enzyme Production and Characterization

A trypsin-like protease was purified from spent culture medium of oral pathogen Porphyromonaa gingiva-Lis by chromatography on columns of DEAE-Sepharose, gel filtration on Sephadex 6-100, and chromatofocusing on PBE-94.Purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 55,OOO.Purified protease hydrolyzed type I, 111, IV, and V collagen from human placenta, and type I collagen from rat tail and calf skin, but did not hydrolyze type I1 collagen from chicken sternal cartilage.The purified enzyme also hydrolyzed the C3 component of complement, fibrinogen, fibronectin, alantitrypsin, aZ-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-~-leucylamido(4-guanidinolbutane (E-W), leupeptin, antipain, salivary histidine-rich protein ( H R P -S ) , 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 €? gingivalis involved in invasion and tissue destruction.Although both qualitative and quantitative differences exist in the microbiota of the adult periodontitis in different patients, the prevalence of Porphyromonas gingivalis in adult periodontitis implicates this organism as an important etiologic agent in diseased state (1-5).Initiation of the disease process and subsequent progression is thought to be dependent on the interaction between host factors and bacterial virulence factors which include the processes of access to host tissue, adherence and colonization, and mediation of tissue destruction.Several potential virulence factors of I! gingivalis include fimbriae, hemagglutinin, lipopolysaccharides, and various bacterial enzymes and proteases (5).Tissue destruction associated with periodontal disease may partially be attributed to the effects of proteases which destroy the gingival and periodontal connective tissue.Several different proteases capable of hydrolyzing collagen (-), immunoglobulins (g-ll), iron-binding proteins (12), and complement