Abstract
Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.
Highlights
Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms
In this report we describe the purification and characterization of another cell surface-associated serine proteinase of P. gingivalis with a novel prolyl tripeptidyl peptidase activity but with a surprising primary structure related to dipeptidyl peptidases
Enzyme Localization, Purification, and Initial Characterization—Analysis of amidolytic activity against H-Ala-Phe-PropNA in several fractions of P. gingivalis, HG66, W50, and ATCC 33277, clearly indicated that an enzyme(s) with prolyl tripeptidyl peptidase activity is localized on the cell surface in all strains tested, with Ͻ5% of the total activity being found in the medium regardless of the growth phase of the bacterial culture
Summary
Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. Recent studies have indicated that this periodontopathogen produces at least seven different enzymes belonging to the cysteine and serine catalytic classes of peptidases, among which three cysteine proteinases (gingipains) are predominant (for review, see Ref. 4) Because their structure, function, enzymatic properties, and pathological significance are known, these endopeptidases are the best characterized group of P. gingivalis enzymes. An enzyme referred to as glycylprolyl peptidase (dipeptidyl peptidase IV) was found to be associated with bacterial surfaces [16], and two molecular mass forms of this peptidase have been described [17, 18] This enzyme(s) has been shown to possess the ability to hydrolyze partially degraded type I collagen, releasing the Gly-Pro dipeptide, and it was suggested that, in collaboration with collagenase, dipeptidyl peptidase IV may contribute to the destruction of the periodontal ligament [19]. In addition to this potential pathological function, glycylprolyl peptidase may play a vital role in providing P. gingivalis with dipeptides, which can be transported inside the cell and serve as a source of carbon, nitrogen,
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