Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis.
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 arginine-Sepharose. 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 P1-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 alpha-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, alpha-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.
Potempa
J
J
Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, 31-120 Kraków, Poland. potempa@arches.uga.edu
Mikolajczyk-Pawlinska
J
J
Brassell
D
D
Nelson
D
D
Thøgersen
I B
IB
Enghild
J J
JJ
Travis
J
J
D018829:Adhesins, Bacterial / Q000235:genetics*; D000595:Amino Acid Sequence; D003094:Collagen / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D000080867:Gingipain Cysteine Endopeptidases; D006033:Glycylglycine / Q000494:pharmacology; D006388:Hemagglutinins / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D018829:Adhesins, Bacterial; D015853:Cysteine Proteinase Inhibitors; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D007527:Isoenzymes; D006033:Glycylglycine; D003094:Collagen; D003546:Cysteine Endopeptidases