Comparative Properties of Two Cysteine Proteinases (Gingipains R), the Products of Two Related but Individual Genes ofPorphyromonas gingivalis

Abstract

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginineSepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P 1 -arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of ␣-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, ␣-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.

Activation of Human Prothrombin by Arginine-specific Cysteine Proteinases (Gingipains R) from Porphyromonas gingivalis *

“…lation product is a single protein with a primary structure essentially identical to the catalytic domain of HRgpA (30,31). Despite both a structural similarity and a specificity restricted to Arg-Xaa peptide bonds, HRgpA and RgpB show considerable differences in catalytic potency (26) which is most profoundly manifested in their ability to activate factor X (32) and protein C (33). In addition to activation of these members of the coagulation cascade pathway, it was also shown that RgpB was capable of generating kallikrein from plasma prekallikrein (16).…”

“…From the culture medium of P. gingivalis HG66 we have purified previously two major forms of arginine-specific cysteine proteinases, HRgpA 1 and RgpB, formerly referred to as high molecular mass gingipain R (95-kDa gingipain R1 or HRGP) and 50-kDa gingipain R2 (RGP-2), respectively (24,26). Both of these enzymes are products of two distinct but related genes (27).…”

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

“…The separated proteins were then transferred to BioTrace nitrocellulose membranes (Pall Corporation) and processed at 15 V for 25 min with a Semi-Dry Trans-blot apparatus (Bio-Rad). The blots were probed with gingipain-specific antibodies (Potempa et al, 1998). The secondary antibody used was immunoglobulin G (heavy plus light chains)-horseradish peroxidase conjugate (Zymed Laboratories).…”

Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture

“…Inactivation through autocatalytic processing also occurred during purification under ordinary conditions. We attempted to reversibly inhibit the Clp activity with inhibitors such as EDTA and 4,49-dithiopyridine disulfide (Potempa et al, 1998), but we failed to fully restore the Clp activity by incubation in the activation buffer (data not shown). Another characteristic feature of Clp is that it is easily denatured into insoluble aggregates unless a high ionic strength buffer (0.15 M or higher NaCl) is used.…”