Coemergence of the Amphipathic Helix on Ameloblastin With Mammalian Prismatic Enamel

Abstract

To investigate correlation between the ameloblastin (Ambn) amino acid sequence and the emergence of prismatic enamel, a notable event in the evolution of ectodermal hard tissues, we analyzed Ambn sequences of 53 species for which enamel microstructures have been previously reported. We found that a potential amphipathic helix (AH) within the sequence encoded by exon 5 of Ambn appeared in species with prismatic enamel, with a few exceptions. We studied this correlation by investigating synthetic peptides from different species. A blue shift in fluorescence spectroscopy suggested that the peptides derived from mammalian Ambn interacted with liposomes. A downward shift at 222 nm in circular dichroism spectroscopy of the peptides in the presence of liposomes suggested that the peptides of mammals with prismatic enamel underwent a transition from disordered to helical structure. The peptides of species without prismatic enamel did not show similar secondary structural changes in the presence of liposomes. Peptides of mammals with prismatic enamel caused liposome leakage and inhibited LS8 and ALC cell spreading regulated by full-length Ambn. RT-PCR showed that AH is involved in Ambn’s regulation of cell polarization genes; Vangl2, Vangl1, Prickle1, ROCK1, ROCK2 and Par3. Our comprehensive sequence analysis clearly demonstrates that AH motif is closely related to the emergence of enamel prismatic structure, providing insight into the evolution of complex enamel microstructure. We speculate that the AH motif evolved in mammals to interact with cell membrane, triggering signaling pathways required for specific changes in cell morphology associated with the formation of enamel prismatic structure.

The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane

“…Remarkably, the N-terminal region of Ambn exon 5 also includes an evolutionarily conserved Y/F-x-x-Y/L/F-x-Y/F motif that mediates self-assembly in Amel, Ambn, and other intrinsically disordered proteins [ 27 ] and could conceivably drive Amel–Ambn co-assembly. Collectively, studies uncovering Ambn–membrane [ 7 , 25 , 26 ] and Amel–Ambn [ 28 , 29 , 30 ] interactions support the notion that the N-terminal region of Ambn exon 5 associates both with membranes and with Amel in addition to being involved in Ambn self-assembly.…”

“…This sequence forms an amphipathic helix (AH) in the presence of phospholipid bilayer membrane vesicles in vitro [ 25 ] and binds to ameloblast-like cell plasma membranes [ 7 ]. A comprehensive study of Ambn’s secondary structure across animal species each carrying prismatic, nonprismatic, or no enamel showed that prismatic enamel formation strongly correlates to this AH-forming sequence and its ability to interact with membranes [ 26 ]. Remarkably, the N-terminal region of Ambn exon 5 also includes an evolutionarily conserved Y/F-x-x-Y/L/F-x-Y/F motif that mediates self-assembly in Amel, Ambn, and other intrinsically disordered proteins [ 27 ] and could conceivably drive Amel–Ambn co-assembly.…”

Modeling ameloblast-matrix interactions using 3D cell culture

“…This observation can be the result of loss of AMBN -cell binding, ( Su et al, 2020 ), or AMBN self-assembly ( Wald., et al, 2017 ). AMBN exon 5 encoded region contains the amphipathic helix (AH) cell binding domain that recently was found to be highly conserved among mammals and had a strong evolutionary relationship with enamel prismatic structure ( Su et al, 2022 ). The same region contains the Y/F-x-x-Y/L/F-x-Y/F self-assembly motif, and the disruption of this motif resulted in a perturbed enamel prismatic architecture ( Wald et al, 2017 ).…”

“…This discrepancy could be due to the differences in the height of the secretory stage ameloblasts in fully developed enamel organs compared to the individual cells in 3D culture. We have recently reported that the addition of functional AMBN to LS-8 cells in culture resulted in an upregulation of planar cell polarity protein Vangl2 (2.8 times) and cell polarity protein Par-3 (3.8 times) when compared to controls (heat denatured AMBN and AMBN Δ5) ( Su et al, 2022 ). Vangl2 planar cell polarity protein is highly expressed particularly in the Tomes’ processes of secretory stage ameloblasts ( Nishikawa and Kawamoto, 2012 ).…”

“…Using the immunofluorescent localization of peptides, it was observed that the AH domain could be specifically localized to ALC cell processes ( Su et al, 2020 ). Furthermore, in ameloblast cell lines, the gene expression levels of cell polarity and planar cell polarity proteins were upregulated in the presence of ameloblastin ( Su et al, 2022 ). In the present study, given the rapid in vivo processing of ameloblastin, the newly expressed recombinant 17 kDa ameloblastin containing the exon 5 encoded region was tested in 3D on HAT-7 cells to examine the potential in vivo significance of ameloblastin-cell interactions.…”