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.