Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide

Abstract

Previously, we have shown that serine-16 phosphorylation in native full-length porcine amelogenin (P173) and the Leucine-Rich Amelogenin Peptide (LRAP(+P)), an alternative amelogenin splice product, affects protein assembly and mineralization in vitro. Notably, P173 and LRAP(+P) stabilize amorphous calcium phosphate (ACP) and inhibit hydroxyapatite (HA) formation, while non-phosphorylated counterparts (rP172, LRAP(−P)) guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases. To test this hypothesis, we have utilized Fourier transform infrared spectroscopy (FTIR) to determine the secondary structure of LRAP(−P) and LRAP(+P) in the absence/presence of calcium and selected mineral phases relevant to amelogenesis; i.e., hydroxyapatite (HA: an enamel crystal prototype) and (ACP: an enamel crystal precursor phase). Aqueous solutions of LRAP(−P) or LRAP(+P) were prepared with or without 7.5 mM of CaCl2 at pH 7.4. FTIR spectra of each solution were obtained using attenuated total reflectance, and amide-I peaks were analyzed to provide secondary structure information. Secondary structures of LRAP(+P) and LRAP(−P) were similarly assessed following incubation with suspensions of HA and pyrophosphate-stabilized ACP. Amide I spectra of LRAP(−P) and LRAP(+P) were found to be distinct from each other in all cases. Spectra analyses showed that LRAP(−P) is comprised mostly of random coil and β-sheet, while LRAP(+P) exhibits more β-sheet and α-helix with little random coil. With added Ca, the random coil content increased in LRAP(−P), while LRAP(+P) exhibited a decrease in α-helix components. Incubation of LRAP(−P) with HA or ACP resulted in comparable increases in β-sheet structure. Notably, however, LRAP(+P) secondary structure was more affected by ACP, primarily showing an increase in β-sheet structure, compared to that observed with added HA. These collective findings indicate that phosphorylation induces unique secondary structural changes that may enhance the functional capacity of native phosphorylated amelogenins like LRAP to stabilize an ACP precursor phase during early stages of enamel mineral formation.

Phosphorylated and Non-phosphorylated Leucine Rich Amelogenin Peptide Differentially Affect Ameloblast Mineralization

“…Therefore, its topical application can be envisioned for a future repair of enamel lesions. Furthermore, our ex vivo observations on LRAP(+P) correlate with the recent in vitro findings by Yamazaki and colleagues on the native phosphylated amelogenins during the early stages of enamel formation (Yamazaki et al, 2017 ). Unraveling the signaling pathways underlying this action is therefore mandatory for a potential use of this peptide as early treatment of inborn disorders of enamel.…”

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

“…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 three structural proteins amelogenin (AMELX), enamelin (ENAM), and ameloblastin (AMBN), and the two matrix proteases matrix-metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are well studied, and their posttranslational modifications impact HAp formation and enamel health (Chun et al, 2010; Wiedemann-Bidlack et al, 2011; Yamakoshi, 2011; Bartlett, 2013; Hu et al, 2014; Lacruz et al, 2017; Yamazaki et al, 2017; Yan et al, 2017; Le Norcy et al, 2018). The spatial organization and timing of protein deposition, posttranslational modification and removal is tightly regulated to guide mineralization.…”

“…The spatial organization and timing of protein deposition, posttranslational modification and removal is tightly regulated to guide mineralization. Yet, 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).…”

Tooth Enamel and Its Dynamic Protein Matrix

“…One needs to keep in mind though, that amelogenin is a relatively small molecule, yet, highly concentrated in the extracellular matrix. It has been shown that this single phosphorylation on serine-16 is of critical importance for mineralization and plays a key role for the regulation of mineral phase and transition from amorphous calcium phosphate to hydroxyapatite [ 69 , 70 , 71 , 72 , 73 ]. The most abundant alternative splice product of amelogenin is the Leucine Rich Amelogenin Peptide (LRAP), with proposed roles in enamel formation, as cell signaling molecule or in the regulation of crystal morphology [ 67 , 74 , 75 , 76 , 77 , 78 , 79 ].…”

“…The most abundant alternative splice product of amelogenin is the Leucine Rich Amelogenin Peptide (LRAP), with proposed roles in enamel formation, as cell signaling molecule or in the regulation of crystal morphology [ 67 , 74 , 75 , 76 , 77 , 78 , 79 ]. 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 ].…”