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

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...

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Autores principales: Yamazaki, Hajime, Beniash, Elia, Yamakoshi, Yasuo, Simmer, James P., Margolis, Henry C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489624/
https://www.ncbi.nlm.nih.gov/pubmed/28706493
http://dx.doi.org/10.3389/fphys.2017.00450
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author Yamazaki, Hajime
Beniash, Elia
Yamakoshi, Yasuo
Simmer, James P.
Margolis, Henry C.
author_facet Yamazaki, Hajime
Beniash, Elia
Yamakoshi, Yasuo
Simmer, James P.
Margolis, Henry C.
author_sort Yamazaki, Hajime
collection PubMed
description 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 CaCl(2) 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.
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spelling pubmed-54896242017-07-13 Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide Yamazaki, Hajime Beniash, Elia Yamakoshi, Yasuo Simmer, James P. Margolis, Henry C. Front Physiol Physiology 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 CaCl(2) 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. Frontiers Media S.A. 2017-06-29 /pmc/articles/PMC5489624/ /pubmed/28706493 http://dx.doi.org/10.3389/fphys.2017.00450 Text en Copyright © 2017 Yamazaki, Beniash, Yamakoshi, Simmer and Margolis. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Physiology
Yamazaki, Hajime
Beniash, Elia
Yamakoshi, Yasuo
Simmer, James P.
Margolis, Henry C.
Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide
title Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide
title_full Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide
title_fullStr Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide
title_full_unstemmed Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide
title_short Protein Phosphorylation and Mineral Binding Affect the Secondary Structure of the Leucine-Rich Amelogenin Peptide
title_sort protein phosphorylation and mineral binding affect the secondary structure of the leucine-rich amelogenin peptide
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489624/
https://www.ncbi.nlm.nih.gov/pubmed/28706493
http://dx.doi.org/10.3389/fphys.2017.00450
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