title: Identification of Lys-Pro-Gln as a novel cleavage site specificity of saliva-associated proteases.
abstract: The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins. The proteolytic cleavages in the basic proline-rich proteins occurred preferentially after a Gln residue with predominant specificity for the tripeptide Xaa-Pro-Gln, where Xaa in the P(3) position was mostly represented by Lys. Using the synthetic substrates Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA, the overall K(m) values were determined to be 97 +/- 7.7 and 611 +/- 28 microm, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P(2) and P(3) on protease recognition. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity was most effectively inhibited by antipain and 4-(2-aminoethyl) benzenesulfonyl fluoride, was metal ion-dependent, and not inhibited by cysteine protease inhibitors. A systematic evaluation of enzyme activities in various exocrine and nonexocrine contributors to whole saliva revealed that the glutamine endoprotease is derived from dental plaque and likely microbial in origin. The P(1) site being occupied by a Gln residue is a nonarchetype with respect to known proteases and indicates the presence of novel glutamine-specific endoprotease(s) in oral fluid.
authors.affiliation: Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA. helmer@bu.edu
authors.forename: Eva J
authors.initials: EJ
authors.lastname: Helmerhorst
authors.forename: Xiuli
authors.initials: X
authors.lastname: Sun
authors.forename: Erdjan
authors.initials: E
authors.lastname: Salih
authors.forename: Frank G
authors.initials: FG
authors.lastname: Oppenheim
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009055:Mouth / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D012463:Saliva / Q000201:enzymology*; D012471:Salivary Proteins and Peptides / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D012471:Salivary Proteins and Peptides; D010447:Peptide Hydrolases