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J. Biol. Chem., Vol. 283, Issue 29, 19957-19966, July 18, 2008

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

From the Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118

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 K_m 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.

Received for publication, October 4, 2007 , and in revised form, April 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants DE05672, DE07652, DE18132, and DE16699. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Boston University Goldman School of Dental Medicine, Dept. of Periodontology and Oral Biology, 700 Albany St., W-201, Boston, MA 02118. Fax: 617-638-4924; E-mail: helmer{at}bu.edu.

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