Complete Nucleotide Sequence of a Gene prtR of Porphyromonas gingivalis W50 Encoding a 132-kDa Protein That Contains an Arginine-Specific Thiol Endopeptidase Domain and a Hemagglutinin Domain

Kirszbaum, L.; Sotiropoulos, C; Jackson, C.; Cleal, Steven M.; Slakeski, Nada; Reynolds, Eric C.

doi:10.1006/bbrc.1995.1205

Cited by 43 publications

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“…The PrtR45 Arg-specific proteinase component of the PrtR complex has the same characteristics and N-terminal sequence as the 50 kDa Arg-specific proteinase identified in the culture supernatant of P. gingivalis H66 by Chen et al (1992) designated Arg-gingipain and also has the same characteristics and N-terminal sequence as the 44 kDa enzyme purified from the culture supernatant of P. gingivalis 381 by Okamoto et al (1995) designated Argingipain. The difference in size between the 44/45 and 50 kDa proteinases probably results from C-terminal truncation of the 50 kDa enzyme as suggested previously (Kirszbaum et al, 1995).…”

Section: Discussionsupporting

confidence: 66%

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

1997

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Porphyromonas gingiwalis has been associated with the development of adult periodontitis and cysteine proteinases with trypsin-like specificity have been implicated as major virulence factors. We have extracted the major cellassociated trypsin-like proteolytic activity of P. gingiwalis W50 using mild sonication. Anion-exchange and gel-f iltration FPLC of the sonicate revealed that Arg-and Lys-specific proteinase activity was associated with a 300 kDa complex which could be dissociated into seven bands (48,45,44,39,27,17 and 15 kDa) by SDS-PAGE with the 4 4 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on ArgSepharose the majority of the complex eluted from the affinity column as an undissociated complex. However, a small amount dissociated such that the Lys-and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa protein of the complex was purified by further anion-exchange FPLC in the presence of octylpD-glucopyranoside and was shown to be an Arg-specif ic, thiol-activated, calcium-stabilized cysteine proteinase. The 48 kDa protein was also further purified in a similar fashion and shown to be a Lys-specific cysteine proteinase that was not inhibited by EDTA. The two 44 kDa and the 39,27,17 and 15 kDa proteins of the complex exhibit amino acid sequence homology and are proposed to be haemagglutinindadhesins. The 45 kDa Arg-specif ic proteinase and one of the 44 kDa adhesins as well as the 15,17 and 27 kDa adhesins are processed from the single polyprotein encoded by the gene designated prtR, with all proteins preceded by an Arg or Lys residue within the polyprotein. Similarly, the 48 kDa Lys-specific proteinase, the 39 and 15 kDa adhesins as well as the other 44 kDa adhesin of the 300 kDa complex are encoded by a single gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. The 39,15 and 44 kDa adhesins of PrtK all exhibit high homology with the 44,15,17 and 27 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, designated PrtR-PrtK, is therefore composed of the two gene products, the mature PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteolytically processed (most likely autolytically) to release proteinase and adhesin domains that remain non-covalently associated. The fully processed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 and PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtRl5, PrtRl7, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingiwalis On: Sat, 12 May 2018 21:46:32 P. S. BHOGAL, N. SLAKESKI a n d E. C. REYNOLDS involved in the evasion of host defence and in the assimilation of haem and peptides.

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Section: Discussionsupporting

confidence: 66%

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

1997

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“…A common finding in studies of these enzymes has been the association of hemagglutinating activity with the purified protease (35,40,41). A molecular explanation for this association has emerged recently following the purification and characterization of the major arginine-specific and lysine-specific protease activities and the cloning and sequence analysis of the corresponding genes from P. gingivalis W50 (1,23), HG66 (39), and 381 (36,37).…”

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confidence: 99%

The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin

et al. 1997

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The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, ␣, ␤, and ␥) and is processed to a heterodimeric protease (RI) which comprises the ␣ and ␤ components in a noncovalent association. The ␣ component contains the protease active site, whereas the ␤ component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI ␤ component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an M r 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the ␤ domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI ␤ component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B 12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter ؊1 ), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine-and lysine-specific protease activities than the parent strain, indicating that there may be a regulatory relationship between tla and other members of this gene family.Porphyromonas gingivalis, an anaerobic gram-negative rod bacterium, is regarded as an important etiological agent in chronic adult periodontal disease (15, 49). This highly prevalent disease is one of a group of inflammatory conditions which undermine the attachment apparatus of the teeth by destroying the soft connective tissues of the periodontium and the alveolar bone. These destructive processes may be a consequence of direct action of microbial products derived from the subgingival plaque on the adjacent tissues of the periodontium and/or caused by the deregulation of the host's inflammatory response. The extracellular proteases of P. gingivalis have the potential to contribute to both of these mechanisms of tissue destruction via the degradation of a wide range of host macromolecules which are fundamental not only to tissue integrity but also to the control of inflammation (22,47,57).Biochemical characterization of P. gingivalis proteases has demonstrated a complex pattern of enzymes with different molecular...

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“…At least seven different P. gingivalis strains have been used in recent studies describing proteinases (1-3, 5-7, 9-12, 18, 20, 22, 35, 37, 38, 45). Some investigators have purified proteinases from spent culture medium (1,2,5,10,12,18,41,45) or extracted them from cells (6,11,20,22,35,44); others have characterized a cloned gene product (3,9,19,37,38). Moreover, a variety of substrates, activators, and inhibitors have been used in various assays to characterize the enzymes.…”

mentioning

confidence: 99%

“…A number of P. gingivalis genes purportedly encoding proteinases have recently been cloned and sequenced, including rgp-1, prpR1, prtR, prtH, agp, cpgR, prtT, tpr, and prtC (1,3,9,12,19,20,30,36,37,39). To date, the relationships among these genes have been unclear, although some authors have noted a likeness in the rgp-1, prpR1, prtR, agp, and cpgR sequences (1,20,34,42). Except for PrtC, which apparently is a collagenase, the proteases associated with these genes cleave after arginine residues in synthetic substrates.…”

mentioning

confidence: 99%

Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis

et al. 1996

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The cloning and sequencing of the gene encoding porphypain, a cysteine proteinase previously isolated from detergent extracts of the Porphyromonas gingivalis W12 cell surface, are described. The prtP gene encoded a unique protein of 1,732 amino acids, including a putative signal sequence for protein secretion. The predicted molecular mass for the mature protein was 186 kDa, which was close to the observed molecular mass of 180 kDa. There was one copy of prtP in the genomes of seven P. gingivalis strains examined. The gene was located 5 to a region with a high degree of homology to the insertion element IS1126 in P. gingivalis W12. The PrtP protein had regions of high homology to HagA, a hemagglutinin of P. gingivalis, and to several purported proteinases of P. gingivalis that have Arg-X specificity. A detailed comparison of genes encoding the latter and cpgR suggested that rgp-1, prpR1, prtR, agp, cpgR, and possibly prtH were derived from identical genetic loci. Although an rgp-1-like locus was detected in seven P. gingivalis strains by Southern blot analyses, agp and cpgR were not detected, not even in the strains from which they were originally isolated. In addition, at least 20 copies of a repeat region common to PrtP, the Rgp-1-like proteins, and HagA were observed in each of the seven genomes examined. The repeat region hybridization patterns for strains W83 and W50 were very similar, and they were identical for strains 381 and ATCC 33277, providing further evidence that these strains are closely related genetically.

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Complete Nucleotide Sequence of a Gene prtR of Porphyromonas gingivalis W50 Encoding a 132-kDa Protein That Contains an Arginine-Specific Thiol Endopeptidase Domain and a Hemagglutinin Domain

Cited by 43 publications

References 0 publications

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin

Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis

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