Purification and characterization of arginine carboxypeptidase produced by Porphyromonas gingivalis

Abstract

Arginine carboxypeptidase was isolated from the cytoplasm of Porphyromonas gingivalis 381 and purified by DEAE-Sephacel column chromatography, followed by high-performance liquid chromatography on DEAE-5PW and TSK G2000SW(XL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of three major bands at 42, 33, and 32 kDa with identical N-terminal sequences. By Western blotting analysis and immunoelectron microscopy, the arginine carboxypeptidase was found to be widely distributed in the cytoplasm and on the surface of the outer membrane. The open reading frame corresponding to the N-terminal amino acids of the arginine carboxypeptidase was detected by a search of the sequence of the P. gingivalis W83 genome. This sequence showed homology with mammalian carboxypeptidases (M, N, and E/H) and included a zinc-binding region signature, suggesting that the enzyme is a member of the zinc carboxypeptidase family. The purified enzyme was inhibited by EGTA, o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and some metal ions, such as Cu(2+), Zn(2+), and Cd(2+). On the other hand, Co(2+) activated the enzyme. The enzyme released arginine and/or lysine from biologically active peptides containing these amino acids at the C terminus but did not cleave substrates when proline was present at the penultimate position. These results indicate that the arginine carboxypeptidase produced by P. gingivalis is an exo type of metallocarboxypeptidase. This enzyme may function to release arginine in collaboration with an arginine aminopeptidase, e.g., Arg-gingipain, to obtain specific amino acids from host tissues during the growth of P. gingivalis.

FIG. 1.

Western blotting analysis of cell fractions of P. gingivalis. Western blotting was performed with an antibody against RCP (1:1,000 dilution). Following incubation with the antibody, the nitrocellulose membrane was reacted with a goat anti-rabbit antibody conjugated to horseradish peroxidase (1:3,000 dilution). Lanes: 1, periplasm (30 μg); 2, cytoplasm (30 μg); 3, cytoplasmic membrane-enriched cell envelope (30 μg); 4, outer membrane-enriched cell envelope (30 μg); 5, purified RCP (14 μg). The values on the left are molecular sizes in kilodaltons.

FIG. 2.

Immunogold localization of P. gingivalis RCP. Thin-section electron photomicrographs of P. gingivalis cells. P. gingivalis cells were incubated with preimmune rabbit serum (A) or anti-RCP serum (B), followed by anti-rabbit IgG conjugated to 10-nm gold particles. Note that some of the immunogold-labeled colloid was observed in both the cytoplasm and the surface-associated compartments of P. gingivalis cells. Bars, 0.25 μm.

FIG. 3.

Second gel filtration pattern of the RCP through a TSK G2000SW_XL gel HPLC column. One of the enzyme active fractions from the HPLC on DEAE-5PW that contained no trypsin-like enzyme was subjected to gel filtration on an HPLC column packed with TSK G2000SW_XL gel that had been equilibrated with 10 mM Tris-HCl buffer (pH 8.2) containing 0.15 M NaCl. The enzyme active fraction was collected, concentrated, and gel filtered again through the same HPLC column. By this repeated gel filtration, the fraction containing RCP activity was eluted as a single, symmetric peak at a position corresponding to an estimated molecular mass of 35 kDa. Symbols: •, protein content; ○, RCP activity

FIG. 4.

SDS-PAGE pattern of the purified RCP from the cytoplasm of P. gingivalis. Lanes: 1, molecular size markers (β-galactosidase [116 kDa], BSA [66 kDa], aldolase [42 kDa], carbonic anhydrase [30 kDa], and myoglobin [17 kDa]); 2, purified enzyme (7 μg of protein was applied). The values shown are molecular sizes in kilodaltons.

FIG. 5.

Suspected RCP sequence from the unfinished P. gingivalis W83 genome. The ORF of the DNA sequence and the deduced amino acid sequence are shown. The underlined amino acid sequence was determined by sequencing the N terminus of the isolated protein. The consensus signature sequence of the zinc-binding region conserved in zinc carboxypeptidases is doubly underlined. Potential N-linked glycosylation sites are boxed.