title: Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.
abstract: Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blotting, and cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with MMP-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. MMP-20 cleaved each peptide exactly at the sites corresponding to ameloblastin cleavages catalyzed in vivo. Klk4 cleaved ameloblastin and the fluorescent peptides at sites not observed in vivo, and cleaved at only a single correct site: before Leu(171). We conclude that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis, and we present a hypothesis about the sequence of ameloblastin cleavages.
authors.affiliation: Department of Biologic and Materials Sciences, University of Michigan, School of Dentistry, 1011 North University, Ann Arbor, MI 48109-1078, USA.
authors.forename: Y-H P
authors.initials: YH
authors.lastname: Chun
authors.forename: Y
authors.initials: Y
authors.lastname: Yamakoshi
authors.forename: F
authors.initials: F
authors.lastname: Yamakoshi
authors.forename: M
authors.initials: M
authors.lastname: Fukae
authors.forename: J C-C
authors.initials: JC
authors.lastname: Hu
authors.forename: J D
authors.initials: JD
authors.lastname: Bartlett
authors.forename: J P
authors.initials: JP
authors.lastname: Simmer
mesh_terms: D000566:Amelogenesis / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003746:Dental Enamel Proteins / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D013058:Mass Spectrometry; D053519:Matrix Metalloproteinase 20 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D034421:Sus scrofa
chemical_list: D003746:Dental Enamel Proteins; D010455:Peptides; D011994:Recombinant Proteins; C490891:ameloblastin protein, Sus scrofa; D007610:Kallikreins; C120801:kallikrein 4; D053519:Matrix Metalloproteinase 20
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