Lys-Pro-Gln, a novel cleavage site specificity of saliva-associated proteases

Proteolytic activities in whole saliva (WS) fluid have been well documented and are believed to be in part attributable to the non-glandular contributors to whole saliva. In our previous studies investigating the whole saliva peptidome, a remarkable and consistent cleavage activity was observed C-terminal to glutamine residues, which are abundantly present in proline-rich proteins (PRPs). In basic PRPs, the tripeptide Lys-Pro-Gln appeared to be a preferred cleavage site. Aim: To determine the kinetic parameters of the apparent glutamine endoprotease activity in WS using selected synthetic substrates. Methods: Two paranitroanilide (-pNA) derivitized peptides, Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA were synthesized, and the kinetics of their hydrolysis in pooled WS supernatant was followed spectrophotometrically at 405 nm at 37^oC. Using the Lys-Pro-Gln-pNA substrate, the pH optimum for enzymatic activity and inhibitor profiles were also determined. Results: The overall K_m values for Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA hydrolysis in WS supernatant were 97 ± 7.7 µM and 611 ± 28 µM, respectively, confirming glutamine endoprotease activity in WS and the importance of the amino acids in positions P₂ and P₃. The V_max values for both substrates were similar (1.8 µmol.l^-1.min^-1) indicating that the same enzymes could be involved in the proteolysis of both substrates. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity could be partly inhibited by serine- and metallo protease inhibitors, but not by cysteine protease inhibitors. Conclusion: The present study, employing artificial enzymatic substrates, supports our previous observation that glutamine endoproteases are characteristic enzymes among the proteases present in WS. Supported by NIH/NIDCR Grants DE05672, DE07652, and DE16699.