Open AccessMmp-20 and Klk4 Cleavage Site Preferences for Amelogenin Sequences
Author(s) -
Takatoshi Nagano,
Ayako Kakegawa,
Yasuo Yamakoshi,
Shunji Tsuchiya,
Jan C.C. Hu,
Kazuhiro Gomi,
Takashi Arai,
John D. Bartlett,
James P. Simmer
Publication year - 2009
Publication title -
journal of dental research
Language(s) - English
Resource type - Journals
eISSN - 1544-0591
pISSN - 0022-0345
DOI - 10.1177/0022034509342694
Subject(s) - amelogenin , chemistry , cleavage (geology) , proteases , biochemistry , ameloblast , enamel paint , microbiology and biotechnology , enzyme , biology , gene , dentistry , medicine , paleontology , fracture (geology)
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 Pro(162), Ser(148), His(62), Ala(63), and Trp(45). 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 Pro(45) and Pro(40), producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His(62). We propose that Mmp-20 alone processes amelogenin during the secretory stage.