Identification of Lys-Pro-Gln as a Novel Cleavage Site Specificity of Saliva-associated Proteases

• Published in: The Journal of biological chemistry Vol. 283; no. 29; pp. 19957 - 19966
• Main Authors: Helmerhorst, Eva J., Sun, Xiuli, Salih, Erdjan, Oppenheim, Frank G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.07.2008; American Society for Biochemistry and Molecular Biology
• Subjects: Adult; Amino Acid Sequence; Chromatography, High Pressure Liquid; Humans; Hydrolysis; Kinetics; Molecular Sequence Data; Mouth - enzymology; Peptide Hydrolases - metabolism; Peptides - metabolism; Protein Synthesis, Post-Translational Modification, and Degradation; Saliva - enzymology; Salivary Proteins and Peptides - metabolism; Substrate Specificity; Tandem Mass Spectrometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M708282200

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 P3 position was mostly represented by Lys. Using the synthetic substrates Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA, the overall Km values were determined to be 97 ± 7.7 and 611 ± 28 μm, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P2 and P3 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 P1 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.