Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin, which is known to bind to hydroxyapatite. However, the role of LRAP in enamel crystal growth and its binding to enamel crystals remain unclear.  Objective: To identify the role of the C-terminus of LRAP in binding onto the surface of the synthetic 3% carbonated hydroxyapatite (CAP).  Methods: Recombinant human LRAP (rhLRAP58) and C-terminal truncated LRAP (rhLRAP47) were produced in E. coli, purified by affinity chromatography and reverse-phase HPLC. These peptides were characterized using SDS-PAGE, Western analysis and reconfirmed with mass spectrometry.  The C-terminus of LRAP (STDKTKREEVD) was synthesized by GenScript (> 96.5% purity).  CAP was synthesized and characterized by X-ray diffraction, FT-IR spectroscopy, and specific surface area analysis (74.68 ± 0.31 m²/g).  rhLRAP58 and rhLRAP47 were incubated with CAP in 20 mM of Tris/Cl, pH 7.5 for 2 h at 25 °C.  Concurrently, rhLRAP58 and rhLRAP47 were also incubated with CAP that had been pre-treated with the synthetic C-terminal fragments.  The bound proteins were visualized on SDS-PAGE and quantified using Bradford method.  Statistical analysis (unpaired t-test) was conducted for triplicates per reaction.  Results: The amount of bound rhLRAP58 (23.52 ± 2.98 µg/ mg CAP) was significantly greater than that of bound rhLRAP47 (2.67 ± 1.13 µg/ mg CAP), showing increased binding by more than 88%. The pre-treatment of C-terminal fragments to CAP significantly reduced the binding of rhLRAP58 (5.04 ± 1.54 µg/ mg CAP) by 79%, but it did not greatly affect the binding of rhLRAP47 (2.58 ± 0.80 µg/ mg CAP) as compared to the untreated CAP substrate.  Conclusions: rhLRAP58 binds to CAP surface.  This specific protein/apatite interaction is inhibited by the synthetic charged C-terminus, providing strong evidence that rhLRAP58 binds CAP through the C-terminal domain.

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