MODIFICATION OF CYSTATIN-C ACTIVITY BY BACTERIAL PROTEINASES AND NEUTROPHIL ELASTASE IN PERIODONTITIS

Aim-To study the interaction between the human cysteine proteinase inh ibitor, cystatin C, and proteinases of periodontitis associated bacter ia. Methods-Gingival crevicular fluid samples were collected from disc rete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cy statin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinob acillus actinomycetemcomitans was studied by measuring inhibition of e nzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-term inal sequence analysis of cystatin C products resulting from the inter actions. Results-Gingival crevicular fluid contained cystatin C at a c oncentration of -15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, release d cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal regio n of cystatin C at physiological pH values. The modified cystatin C re sulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (K-i 5 nM) than t hat of full length cystatin C. A 50 kDa thiol stimulated proteinase, g ingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitor y activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10- bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. Conclusions-The physiological control of cathepsin B ac tivity is impeded in periodontitis, owing to the release of proteinase s from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence .