cysteinylglycine and Periodontitis

The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, gamma-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (k cat/Km of 8.2 microm(-1) min(-1)) not l-Leu-p-NA (k cat/Km of 1.1 microm(-1) min(-1)). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, gamma-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.

Topics: Amino Acid Sequence; Bacterial Proteins; Chromatography, High Pressure Liquid; Dipeptidases; Dipeptides; Escherichia coli; gamma-Glutamyltransferase; Genome, Bacterial; Glutathione; Hydrogen Sulfide; Hydrolysis; Leucyl Aminopeptidase; Periodontitis; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Treponema denticola

Recent studies describe the potential use of biochemical markers in the evaluation of the severity of periodontitis; moreover, patients suffering from periodontitis frequently complain of halitosis (breath malodour), mainly depending on volatile compounds (e.g. hydrogen sulphide, methyl mercaptan, etc.) produced by anaerobic metabolism of oral bacteria and involving sulphur-containing amino acids. In this study, salivary sulphur compounds, such as cysteine, cysteinylglycine and glutathione and some markers of cellular damage (lactate dehydrogenase and aspartate amino transferase), were measured in periodontitis patients and correlated with the periodontal probing pocket's depth.. Twenty-two periodontitis patients and forty control subjects were studied for the salivary activities of lactate dehydrogenase and aspartate aminotransferase and cysteine, cysteinylglycine and glutathione concentrations. The periodontitis patients were divided into two subgroups based on the severity of periodontal disease, expressed as median periodontal probing pocket depth (> or <5 mm). Enzyme activities were measured by using an automated clinical analyzer; cysteine, cysteinylglycine and glutathione concentrations were measured by HPLC equipped with fluorescence detector.. A statistically significant increase of the salivary parameters level (cysteine, cysteinylglycine, glutathione, aspartate aminotransferase and lactate dehydrogenase) was found in the patient subgroup with periodontal probing pocket depth >5 mm, the salivary cysteine concentrations showing the most significant correlation.. Salivary cysteine, a direct precursor of hydrogen sulphide, could be considered reliable markers for the oral tissue damage severity in periodontitis patients.

Topics: Adult; Aged; Aspartate Aminotransferases; Biomarkers; Cysteine; Dipeptides; Female; Glutathione; Humans; L-Lactate Dehydrogenase; Male; Middle Aged; Periodontal Pocket; Periodontitis; Saliva; Sulfhydryl Compounds