Identification of a novel proteinase (ameloprotease-I) responsible for the complete degradation of amelogenin during enamel maturation.
During enamel formation the proteins of the extracellular matrix, particularly amelogenins, are removed prior to maturation. In order to investigate this process and to improve our understanding of the function of proteinases during enamel maturation, proteinase fractions were isolated from developing pig enamel and assayed for proteolytic activity in vitro. A recombinant murine amelogenin, M179, was used as a substrate. Two major groups of enamel proteinases were defined as high-molecular-mass ['high-molecular-weight' in Moradian-Oldak, Simmer, Sarte, Zeichner-David and Fincham (1994) Arch. Oral Biol.39, 647-656] and low-molecular-mass proteinases. Here we report the characterization of one of the proteinases present in the low-molecular-mass group. We demonstrate that this proteinase is a serine proteinase capable of degradation of M179 following cleavage of the tyrosine-rich amelogenin polypeptide from the N-terminal region. A partial N-terminal sequence of the proteinase was obtained (LPHVPHRIPPGYGRPXTXNEEGXNPYFXFFXXHG). An anti-peptide antibody directed against a synthetic peptide corresponding to the first 14 amino acids of the above sequence was produced. The presence of the proteinase in the acetic acid extract was confirmed by Western blotting. Searching using the amino acid sequence determined in this study showed it to be also present in the 32 kDa and 89 kDa enamelin proteins reported by Fukae, Tanabe, Murakami and Tohi [(1996) Adv. Dent. Res., in the press]. We therefore identify the 32 kDa enamelin as an enamel proteinase ('ameloprotease-I') which is responsible for amelogenin degradation in maturing enamel. We propose that the 89 kDa enamelin is a precursor of ameloprotease-I, the first enamel protein for which a function has been defined.
Moradian-Oldak
J
J
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles 90033, USA.
Leung
W
W
Simmer
J P
JP
Zeichner-David
M
M
Fincham
A G
AG
D053523:Amelogenin; D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D003743:Dental Enamel / Q000254:growth & development* / Q000378:metabolism*; D003746:Dental Enamel Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D053523:Amelogenin; D003746:Dental Enamel Proteins; D012697:Serine Endopeptidases; C101855:ameloprotease I; D000645:Ammonium Sulfate