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Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity.

ABSTRACT: The prtT gene, coding for trypsinlike proteolytic activity, has been isolated from Porphyromonas gingivalis ATCC 53977. This gene is present immediately downstream from the sod gene on a 5.9-kb DNA fragment from the organism isolated in Escherichia coli. The complete nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the enzyme corresponds to a 53.9-kDa protein with an estimated pI of 11.85. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography also indicated a similar molecular size for the protease. The enzyme was purified to near homogeneity following anion-exchange and gel-filtration chromatography. The purified enzyme also exhibited a single protein species with a size of approximately 53 kDa. Enzyme activity was strongly dependent upon the presence of reducing agents (dithiothreitol, cysteine, and 2-mercaptoethanol) and was also stimulated in the presence of calcium ions. A comparison of the properties of the prtT gene product with comparable parameters of proteases previously purified from different strains of P. gingivalis suggested that the cloned protease represents a previously uncharacterized enzyme.

SUBMITTER: Otogoto J

PROVIDER: S-EPMC302695 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity.

Otogoto J J Kuramitsu H K HK

Infection and immunity 19930101 1

The prtT gene, coding for trypsinlike proteolytic activity, has been isolated from Porphyromonas gingivalis ATCC 53977. This gene is present immediately downstream from the sod gene on a 5.9-kb DNA fragment from the organism isolated in Escherichia coli. The complete nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the enzyme corresponds to a 53.9-kDa protein with an estimated pI of 11.85. Gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis zym ...[more]

PMID: 8093357

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Project description:Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac]0-2)-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane β-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm. IMPORTANCE Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0-2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.

Dataset Information

Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity.

Publications

Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity.

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