Characterization of LRAP Hydrolytic Peptides and Their Biological Activities

Leucine-rich amelogenin peptide (LRAP) is one of the most abundant alternatively spliced amelogenins.  Similar N- and C-terminal sequences between LRAP and full-length amelogenin suggest that LRAP can be hydrolyzed by MMP-20.  Objective:  To characterize LRAP hydrolytic peptides and their biological activities.  Methods:  Human recombinant LRAP (200 µL of 29.2 µM) was incubated with active MMP-20 (3µL of 8.6 µM) in Tris/HCl (20 mM), pH 7.5 at 37 °C.  At specific times over 3 hrs, aliquots of enzymatic reaction mixtures were collected for SDS-PAGE and mass spectrometric analysis.  Absorbance assay and dynamic light scattering (DLS) were used to analyze the aggregation of LRAP hydrolytic products.  Recombinant N-terminal LRAP and synthetic C-terminal peptide (STDKTKREEVD) of LRAP, representing two initial hydrolytic peptides, were synthesized.  Functional assays were used to study the domains of LRAP that bind carbonate hydroxyapatites, and those that promote dental pulp cell proliferation in vitro.  Results:  MMP-20 sequentially hydrolyzed LRAP at 3 different cleavage sites, initially at AWP/STD, followed by DLT/LEA, and finally PLP/PML.  After digestion at 25 °C and 37 °C, LRAP hydrolytic products assembled into peptide aggregates, ranging from 222 ± 19 to 325 ± 47 nm, respectively.  Binding experiments showed that LRAP interacted with carbonate hydroxyapatites by the C-terminal domain.  However, the N-terminal LRAP, lacking the C-terminus, promoted dental pulp cell proliferation in vitro.  Conclusions:  LRAP was hydrolyzed by MMP-20 at 3 cleavage sites.  The C-terminus of LRAP bind directly to apatites, modulating crystal growth, while the N-terminal LRAP hydrolytic product plays a role in cell signaling to regulate tooth formation.  Supported:  NIH/NIDCR T32-DE07306-09 (to T.L.), P01-DE009859 (to P.D.B.) and R01-DE015821 (to W.L.).