PURIFICATION AND CHARACTERIZATION OF A NOVEL ARGININE-SPECIFIC CYSTEINE PROTEINASE (ARGINGIPAIN) INVOLVED IN THE PATHOGENESIS OF PERIODONTAL-DISEASE FROM THE CULTURE SUPERNATANT OF PORPHYROMONAS-GINGIVALIS

A novel arginine-specific cysteine proteinase, termed ''argingipain,'' was purified from culture supernatants of Porphyromonas gingivalis, a n anaerobe commonly associated with progressive periodontal disease, b y conventional chromatographic techniques. The purified enzyme was fou nd to be composed of a single polypeptide of M(r) similar to 44,000. A nalysis of the enzymatic properties revealed several distinctive featu res for this enzyme. The proteolytic activity is absolutely thiol-depe ndent, but the enzyme also has in part the characteristics of both met allo and serine endopeptidases, as shown by the inhibition of activity by metal chelators, chymostatin, and the chloromethyl ketones of tosy l-L-lysine and tosyl-L-phenylalanine. However, internal protease inhib itors, such as cystatins, tissue inhibitor of metalloproteinases, and alpha(1)-antichymotrypsin, have no effects on the activity, suggesting its evasion from normal host defense systems in vivo. Despite its nar row specificity for synthetic substrates containing Arg in the P1 site and hydrophobic amino acids in the P2 or P3 sites, the enzyme extensi vely degrades collagens (types I and IV) and immunoglobulin G. Most im portant, the enzyme has the ability to disrupt the functions of polymo rphonuclear leukocytes, as shown by its inhibitory effect on the gener ation of active oxygen species from the activated cells. Further, the enzyme is found to be produced by all of the species of P. gingivalis examined, but not by other bacteria. These results suggests that argin gipain plays a key role as a major virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host def ense mechanisms.