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Identification of a novel proteinase (ameloprotease-I) responsible for the complete degradation of amelogenin during enamel maturation.

ABSTRACT: 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.

SUBMITTER: Moradian-Oldak J

PROVIDER: S-EPMC1217718 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:Amelogenin and ameloblastin are 2 extracellular matrix proteins that are essential for the proper development of enamel. We recently reported that amelogenin and ameloblastin colocalized during the secretory stage of enamel formation when nucleation of enamel crystallites occurs. Direct interactions between the 2 proteins have been also demonstrated in our in vitro studies. Here, we explore interactions between their fragments during enamel maturation. We applied in vivo immunofluorescence imaging, quantitative co-localization analysis, and a new FRET (fluorescence resonance energy transfer) technique to demonstrate ameloblastin and amelogenin interaction in the maturing mouse enamel. Using immunochemical analysis of protein samples extracted from 8-d-old (P8) first molars from mice as a model for maturation-stage enamel, we identified the ~17-kDa ameloblastin (Ambn-N) and the TRAP (tyrosine-rich amelogenin peptide) fragments. We used Ambn-N18 and Ambn-M300 antibodies raised against the N-terminal and C-terminal segments of ameloblastin, as well as Amel-FL and Amel-C19 antibodies against full-length recombinant mouse amelogenin (rM179) and C-terminal amelogenin, respectively. In transverse sections, co-localization images of N-terminal fragments of amelogenin and ameloblastin around the prism boundary revealed the "fish net" pattern of the enamel matrix. Using in vivo FRET microscopy, we further demonstrated spatial interactions between amelogenin and ameloblastin N-terminal fragments. In the maturing mouse enamel, the association of these residual protein fragments created a discontinuity between enamel rods, which we suggest is important for support and maintenance of enamel rods and eventual contribution to unique enamel mechanical properties. We present data that support cooperative functions of enamel matrix proteins in mediating the structural hierarchy of enamel and that contribute to our efforts to design and develop enamel biomimetic material.

Project description:We report here a novel biomimetic approach to the regeneration of human enamel. The approach combines the use of inorganic pyrophosphate (PPi) to control the onset and rate of enamel regeneration and the use of leucine-rich amelogenin peptide (LRAP), a nonphosphorylated 56-amino acid alternative splice product of amelogenin, to regulate the shape and orientation of growing enamel crystals. This study builds on our previous findings that show LRAP can effectively guide the formation of ordered arrays of needle-like hydroxyapatite (HA) crystals in vitro and on the known role mineralization inhibitors, like PPi, play in the regulation of mineralized tissue formation. Acid-etched enamel surfaces of extracted human molars, cut perpendicular or parallel to the direction of the enamel rods, were exposed to a PPi-stabilized supersaturated calcium phosphate (CaP) solution containing 0 to 0.06 mg/mL LRAP for 20 h. In the absence of LRAP, PPi inhibition was reversed by the presence of etched enamel surfaces and led to the formation of large, randomly distributed plate-like HA crystals that were weakly attached, regardless of rod orientation. In the presence of 0.04 mg/mL LRAP, however, densely packed mineral layers, comprising bundles of small needle-like HA crystals, formed on etched surfaces that were cut perpendicular to the enamel rods. These crystals were strongly attached, and their arrangement reflected to a significant degree the underlying enamel prism pattern. In contrast, under the same conditions with LRAP, little to no crystal formation was found on enamel surfaces that were cut parallel to the direction of the enamel rods. These results suggest that LRAP preferentially interacts with ab surfaces of mature enamel crystals, inhibiting their directional growth, thus selectively promoting linear growth along the c-axis of enamel crystals. The present findings demonstrate a potential for the development of a new approach to regenerate enamel structure and properties.

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Identification of a novel proteinase (ameloprotease-I) responsible for the complete degradation of amelogenin during enamel maturation.

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