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Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin).

ABSTRACT: A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic techniques. The purified enzyme consisted of three polypeptides (38, 43, and 72 kDa). The protease exhibited specificity for peptide bonds containing phenylalanine and proline at the P1 and P2 positions, respectively, and was classified as a serine protease on the basis of inhibition studies. Naturally occurring protease inhibitors such as alpha1-antitrypsin and alpha1-antichymotrypsin had no effect on enzymatic activity. The enzyme degraded fibronectin, alpha1-antitrypsin, and gelatin while weakly degrading the immunoglobulin G heavy chain and type IV collagen. N-terminal amino acid sequences were determined for the 43- and 72-kDa proteins. On the basis of these sequences, the genes coding for the 43- and 72-kDa proteins were isolated and sequenced. The open reading frame which codes for the 72-kDa protein was designated prtP. This gene consists of 2,169 bp and codes for a protein with an Mr of 77,471. The protein appeared to be composed of a signal peptide region followed by a prosequence and the mature protein domain. The deduced amino acid sequence exhibited similarity with that of the Bacillus subtilis serine protease subtilisin. The deduced properties of the sequence suggest that the 72-kDa protein is a chymotrypsin-like protease. However, the nature and function of the 43-kDa protein have not yet been determined.

SUBMITTER: Ishihara K

PROVIDER: S-EPMC174505 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin).

Ishihara K K Miura T T Kuramitsu H K HK Okuda K K

Infection and immunity 19961201 12

A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic techniques. The purified enzyme consisted of three polypeptides (38, 43, and 72 kDa). The protease exhibited specificity for peptide bonds containing phenylalanine and proline at the P1 and P2 positions, respectively, and was classified as a serine protease on the basis of inhibition studies. Naturally occurring protease inhibitors such as alpha1-antitrypsin and alpha1-antichymotrypsin had no effect ...[more]

PMID: 8945563

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Project description:The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5' to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.

Dataset Information

Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin).

Publications

Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin).

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