Functional analysis of leucine-rich amelogenin peptide and its MMP-20-mediated proteolytic products in tooth formation.

摘要

Amelogenins make up over 90% of the secretory enamel proteins and play a critical role in tooth enamel formation. There are several amelogenin isoforms identified within the enamel extracellular matrix as a result of proteolysis and alternative splicing of the primary amelogenin mRNA transcript. Leucine-rich amelogenin peptide (LRAP) is one of the major alternatively spliced amelogenins, whose function in tooth formation remains unclear. The purpose of this thesis is to explore the role and mechanism of LRAP and its MMP-20-mediated proteolytic products in enamel biomineralization, and their biological effects on odontogenic cell (i.e. dental pulp cell and ameloblast-lineage cell) proliferation and/or differentiation. The purified recombinant LRAP was hydrolyzed by active MMP-20. LRAP proteolytic products were analyzed by mass spectrometry to identify specific cleavage sites. Subsequently, LRAP and its proteolytic products were allowed to interact with synthetic carbonate hydroxyapatites (CAP) in vitro to determine the binding mechanism. After binding on the apatite substrates, these peptides were incubated in supersaturated calcium phosphate solution, similar to secretory stage enamel-like fluid, to study their ability to promote enamel-like crystal growth. In addition, LRAP and its proteolytic fragments were also exogenously added to human dental pulp cell and primary ameloblast-lineage cell cultures to detect the evidence of cell proliferation and/or differentiation. Expressions of regulatory genes and cell-surface receptors associated with these specific pathways were also identified by gene arrays, reverse-transcription PCR and immunohistochemistry. LRAP is a specific substrate for MMP-20 hydrolysis to cleave its C-terminal domain, which was responsible for the binding of LRAP directly onto the surface of CAP by ionic interactions. Unlike its parent amelogenin, LRAP did not function as a structural protein to promote crystal formation in vitro; however, it acted as a cell-signaling molecule to affect odontoblast cell proliferation and ameloblast-lineage cell differentiation by up-regulating and interacting with its cell membrane receptor lysosome-associated membrane glycoprotein 1 (LAMP-1). Therefore, LRAP functions in the early stages of cell development to control odontogenesis and amelogenesis. These are novel findings that allow us for the first time to understand how the alternatively spliced amelogenin protein, LRAP and its MMP-20 hydrolysis products, interact with cells and mineral in the developing tooth. This knowledge is critical for further studies of tooth regeneration and the use of amelogenins in biomineralization.