Amelogenins as Potential Buffers during Secretory-stage Amelogenesis

Abstract

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX -/-) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX -/-mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX -/-mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX -/-mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX -/-mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.

Buffering of protons released by mineral formation during amelogenesis in mice

“…Amelogenins can bind to as many as 10–14 protons per molecule in vitro and could potentially serve as a buffer. A buffering role for amelogenins in situ corroborates with the finding that in amelogenin‐deficient ( Amelx‐ null) mice, formation of minerals in enamel is not reduced but initially even accelerated . In addition, immunohistochemistry showed that secretory ameloblasts in Amelx‐ null mice prematurely expressed AE2a,b protein, suggesting that bicarbonate buffering compensates for the absence of buffering by amelogenins .…”

Fluoride

“…This local hypermineralization in the enamel matrix depletes the local reservoir or free calcium ions and then may also act as a physical barrier, impairing diffusion of ions and peptides, to result in a subsequent band of hypomineralized enamel (Guo et al 2015 ). Therefore, within the matrix, it is possible that a more rapid mineral precipitation in the presence of fl uoride may result in the presence of hypermineralized bands followed by hypomineralized bands in the enamel matrix (Bronckers et al 2009 (Guo et al 2015 ).…”

“…Protons generated during HAP formation in the maturation stage are neutralized by bicarbonate secreted from the apical end of ruffl ed-ended maturation ameloblasts through anion exchangers of the Slc26a family (Jalali et al 2014 ). Transmembrane proteins, including cystic fi brosis transmembrane conductance regulator, anion exchanger-2, carbonic anhydrase-2, and sodium hydrogen exchanger-1, are involved in this pH regulation mechanism (Guo et al 2015 ;Lyaruu et al 2008 ;Alper 2009 ;Concepcion et al 2013 ). In the fl uorosed enamel matrix, retention of amelogenin protein may also buffer pH changes in the matrix, secondary to mineral formation, thereby reducing the acidification of the matrix under ruffl e-ended ameloblasts (Guo et al 2015 ).…”

Understanding Dental Caries

“…This local hypermineralization in the enamel matrix depletes the local reservoir or free calcium ions and then may also act as a physical barrier, impairing diffusion of ions and peptides, to result in a subsequent band of hypomineralized enamel (Guo et al 2015 ). This local hypermineralization in the enamel matrix depletes the local reservoir or free calcium ions and then may also act as a physical barrier, impairing diffusion of ions and peptides, to result in a subsequent band of hypomineralized enamel (Guo et al 2015 ).…”

“…In the presence of fluoride, increased mineral formation may result in local acidification, which is buffered by amelogenin proteins in the enamel matrix at the secretory stage (Guo et al 2015 ). Therefore, regulation of matrix pH by ameloblasts is crucial for sustained crystal growth.…”