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.
Highlights
The strength and stiffness of tooth enamel resemble the properties of some metal alloys and are achieved through the hierarchical arrangement of hydroxyapatite (HAp) mineral crystals within a proteinaceous matrix (Bechtle et al, 2012; Wegst et al, 2015; Yilmaz et al, 2015)
We show spatial co-occurrence of proteins pertaining to key steps during enamel secretion and maturation, and follow the spatial abundance P190 and P173+LRAP and their phosphorylation status on serine 16
In these axes bone and dentin data segregate from enamel samples that were clearly associated with secretion and maturation, while data from erupted enamel sorted with both maturation stage and with locations that include a mix of enamel formation stages and tissues
Summary
The strength and stiffness of tooth enamel resemble the properties of some metal alloys and are achieved through the hierarchical arrangement of hydroxyapatite (HAp) mineral crystals within a proteinaceous matrix (Bechtle et al, 2012; Wegst et al, 2015; Yilmaz et al, 2015). The spatiotemporal pattern of relative protein abundance and proteolytic processing, as well as posttranslational modification status including phosphorylation is not fully resolved for the three classic enamel matrix proteins AMELX, ENAM, AMBN, and their cleavage products, and is less resolved for proteins during this process (Uchida et al, 1991, 1997; Tanabe et al, 1992; Moradian-Oldak, 2012; Bartlett, 2013; Gallon et al, 2013; Mazumder et al, 2016; Yamazaki et al, 2017). LRAP phosphorylation appears to affect mineralization activity of ameloblast cell lines and cultured tooth germs (Wiedemann-Bidlack et al, 2011; Le Norcy et al, 2018)
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