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Mmp-20 and Klk4 Cleavage Site Preferences for Amelogenin Sequences

¹ Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, 1011 N. University, Ann Arbor, MI 48109-1078, USA;
² Department of Periodontics and Endodontics, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan; and
³ Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA

Correspondence: ^* University of Michigan Dental Research Lab, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA, jsimmer{at}umich.edu

Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis? We isolated Mmp-20 and Klk4 from developing pig teeth and used them to digest the tyrosine-rich amelogenin polypeptide (TRAP), the leucine-rich amelogenin protein (LRAP), and 5 fluorescence peptides. We characterized the digestion products by LC-MSMS, SDS-PAGE, and C18 RP-HPLC monitored with fluorescence and UV detectors. Mmp-20 cleaves amelogenin sequences after Pro¹⁶², Ser¹⁴⁸, His⁶², Ala⁶³, and Trp⁴⁵. These cleavages generate all of the major cleavage products that accumulate in porcine secretory-stage enamel: the 23-kDa, 20-kDa, 13-kDa, 11-kDa, and 6-kDa (TRAP) amelogenins. Mmp-20 cleaves LRAP after Pro⁴⁵ and Pro⁴⁰, producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His⁶². We propose that Mmp-20 alone processes amelogenin during the secretory stage.

Key Words: TRAP • LRAP • enamel • tooth • fluorescent peptides

Journal of Dental Research, Vol. 88, No. 9, 823-828 (2009)
DOI: 10.1177/0022034509342694


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