title: Role of 20-kDa amelogenin (P148) phosphorylation in calcium phosphate formation in vitro.
abstract: The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.
authors.affiliation: Department of Biomineralization, The Forsyth Institute, Boston, Massachusetts 02115, USA.
authors.forename: Seo-Young
authors.initials: SY
authors.lastname: Kwak
authors.forename: Felicitas B
authors.initials: FB
authors.lastname: Wiedemann-Bidlack
authors.forename: Elia
authors.initials: E
authors.lastname: Beniash
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamakoshi
authors.forename: James P
authors.initials: JP
authors.lastname: Simmer
authors.forename: Amy
authors.initials: A
authors.lastname: Litman
authors.forename: Henry C
authors.initials: HC
authors.lastname: Margolis
mesh_terms: D053523:Amelogenin / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002130:Calcium Phosphates / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D017550:Spectroscopy, Fourier Transform Infrared; D013552:Swine
chemical_list: D053523:Amelogenin; D002130:Calcium Phosphates; D007477:Ions; C020243:calcium phosphate; D002118:Calcium