Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns

Abstract

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Single-Cell RNA-Sequencing From Mouse Incisor Reveals Dental Epithelial Cell-Type Specific Genes

“…This action enables ameloblasts to deposit the enamel matrix on the dentin–enamel junction ( Bartlett, 2013 ). In the secretory stage, pre-ameloblasts increase in height and differentiate into ameloblasts, which develop Tomes’s processes to secrete enamel matrices such as Ambn , Amelogenin ( Amel ), and Enamelin ( Enam ) ( Green et al, 2019 ). At the end of the secretory stage, ameloblasts lose their Tomes’s processes and produce proteases to degrade and absorb the enamel matrix proteins in the transition stage.…”

Tooth Enamel and Its Dynamic Protein Matrix

“…With an increasing number of enamel matrix components being uncovered, the following questions arise—which proteins are endogenous, which ones exogenous, which ones are essential, how do they contribute to enamel development and when do they play a role for enamel formation as well as function? [ 15 ]. The answers to these questions are key to developing clinical benefits, such as improved diagnosis and repair, to leverage biomimetic approaches to regenerate enamel or to create new materials with the exceptional properties of healthy tooth enamel [ 16 , 17 , 18 ].…”

“…Recent studies have provided evidence that the phosphorylation of LRAP induces structural changes that in turn affect enamel mineral formation [ 69 , 80 ]. Whether and when amelogenin is dephosphorylated during amelogenesis is not well known but the question has been addressed in our previous proteomic analyses [ 15 ].…”

“…Serum albumin and typical enamel proteins, such as ameloblastin, amelogenin X isoform, enamelin and Mmp20, were identified in all rat strains and conditions. Notably, Serpin H1 was exclusively detected in mature enamel from resistant mice (129P3/J), observed also in pig samples ( Figure 4 ) [ 15 ]. Serpin H1 is a member of the serpin family of serine and cysteine protease inhibitors and plays an essential role in collagen synthesis with no inhibitory activity [ 110 ].…”

“…Our recent work aimed at filling this gap in knowledge and discerning changes of the mineralizing enamel matrix in different stages of enamel development and in different locations of the mineralizing tooth. The spatio-temporal pattern of protein expression in developing tooth enamel was obtained through combining a micro-sampling approach with proteomic analyses of erupted and unerupted permanent pig molars [ 15 ].…”