Glutamine-Specific Endoprotease Activity in Human Saliva

Introduction: Human saliva is continuously secreted into the oral cavity from where it reaches various portions of the gastro-intestinal tract. The non-sterile oral environment facilitates substantial proteolytic processing of resident salivary proteins. Objective: In order to gain insight into the saliva associated enzymatic processes, the current study utilized mass spectrometric approaches to define the whole saliva peptidome. Methods: The in vivo generated peptides present in unstimulated oral fluid were isolated by centrifugation over a 5 kD filter and subjected to nano-flow LC-ESI-MS/MS. Results: 182 different peptides were identified which were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins, indicating these are the major contributors to the whole saliva peptidome. Analysis of the proteolytic cleavages in the acid and basic PRPs revealed that these occurred preferentially after a glutamine (Gln) residue with predominant specificity for the tripeptide Xaa-Pro-Gln sequence where Xaa in the P₃ position was almost exclusively represented by Lys, Arg or Pro. The P₁ site being occupied by a Gln residue is a non-archetype with respect to known proteases in eukaryotic systems and indicates the presence of as yet unknown saliva-associated proteolytic glutamine-specific endoprotease(s). Conclusion: This study has identified glutamine endoproteases activity in saliva generating novel bioactive peptides from salivary glutamine-containing proteins with potential implications for oral as well as for gastro-intestinal health. Supported by NIH/NIDCR Grants DE05672, DE07652, DE14950, and DE16699.