COMPARATIVE PROPERTIES OF 2 CYSTEINE PROTEINASES (GINGIPAINS-R), THE PRODUCTS OF 2 RELATED BUT INDIVIDUAL GENES OF PORPHYROMONAS-GINGIVALIS

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen, Two of these enzymes, r eferred to as arginine-specific cysteine proteinases (gingipains R), a re the product of two related genes, Here, we describe the purificatio n of an enzyme translated hom the rgpB/rgp-2 gene (gingipain R2, RGP-S ) and secreted as a single chain protein of 422 residues. The enzyme o ccurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In compa rison to the 95-kDa gingipain R1, a complex of catalytic and hemagglut inin/adhesin domains, RGP-2 showed five times lower proteolytic activi ty, although its activity on various P-1-arginine p-nitroanity, substr ates was generally higher, Gingipains R amidolytic activity, but not g eneral proteolytic activity, was stimulated by glycyl-glycine. However , in cases of limited proteolysis, such as the inactivation of alpha-1 -anti-chymotrypsin, glycyl-glycine potentiated inhibitor cleavage, In contrast, alpha-1-proteinase inhibitor was not inactivated by gingipai ns R and only underwent proteolytic degradation during boiling in redu cing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarl y, native type I collagen was completely resistant to cleavage by ging ipains but readily degraded after denaturation, Together, these data e xplain much of the controversy regarding gingipains structure and subs trate specificity and indicate that these enzymes function as P. gingi valis virulence factors by proteolysis of selected target proteins rat her than random degradation of host connective tissue components.