Truncated amelogenin and LRAP transgenes improve Amelx null mouse enamel

Abstract

Amelogenin is the most abundant enamel protein involved in enamel mineralization. Our goal was to determine whether all three regions of amelogenin (N-terminus, C-terminus, central core) are required for enamel formation. Amelogenin RNA is alternatively spliced, resulting in at least 16 different amelogenin isoforms in mice, with M180 and LRAP expressed most abundantly. Soon after secretion by ameloblasts, M180 is cleaved by MMP20 resulting in C-terminal truncated (CTRNC) amelogenin. We aimed to determine whether the 2 transgenes (Tg), LRAP and CTRNC together, can improve LRAPTg/Amelx−/− and CTRNCTg/Amelx−/− enamel thickness and prism organization, which were not rescued in Amelx−/− enamel. We generated CTRNCTg/LRAPTg/Amelx−/− mice and analyzed developing and mature incisor and molar enamel histologically, by microCT, SEM and microhardness testing. CTRNCTg and LRAPTg overexpression together significantly improved the enamel phenotype of LRAPTg/Amelx−/− and CTRNCTg/Amelx−/− mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. We determined that both LRAP and CTRNC, which together express all three regions of the amelogenin protein (N-terminus, C-terminus and hydrophobic core) contribute to the final enamel thickness and prism organization in mice.

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

“…Micro-CT and FE-SEM analyses of the mineral formed by PND1 molar germs after a 9-day culture revealed an increase in enamel volume with LRAP(+P) while it was not increased by LRAP(–P) treatment. Rescue experiments with recombinant plasmid encoding LRAP in amelogenin KO mice, have shown that LRAP contributes to final enamel thickness and prism organization (Gibson et al, 2011 ; Xia et al, 2016 ). It can be speculated from our data, that in these mice LRAP is present under its phosphorylated form.…”

Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

“… Significant differences (p < 0.05) are indicated as follows: w from WT; x from Amelogenin KO; 3o from M180Tg/CTRNCTg/LRAPTg/KO (++); 3t from M180Tg/CTRNCTg/LRAPTg/KO (+); 2o from M180Tg/CTRNCTg/KO (++); 2t from M180Tg/CTRNCTg/KO (+) . a Gibson et al ( 2011 ); b Xia et al ( 2016 ) , c Li et al ( 2008 ) , d Pugach et al ( 2013 ) , e Pugach et al ( 2010 ) , f Chen et al ( 2003 ) , g Gibson et al ( 2009 ) . …”

“…Both recent in vitro and in vivo evidence suggest that CTRNC plays an important role in enamel mineralization (Kwak et al, 2009 , 2011 ; Martinez-Avila et al, 2011 ; Xia et al, 2016 ). In vitro studies have suggested a role of full-length amelogenin in the control of mineral phase, specifically the stabilization of amorphous mineral phases such as amorphous calcium phosphate (ACP), and alignment of forming crystallites (Kwak et al, 2011 ).…”

“…The overexpression of CTRNC Tg and LRAP Tg together improved significantly the enamel phenotype of LRAP Tg /KO and CTRNC Tg /KO mouse enamel, however enamel microhardness was recovered only when M180 Tg was expressed, alone or with LRAP Tg . Expression of LRAP and CTRNC together provides all three regions of the amelogenin protein N-terminus, C-terminus and hydrophobic core further improved the phenotype to reach normal WT enamel thickness and prism organization in the reported mouse model (Chen et al, 2003 ; Pugach et al, 2010 ; Xia et al, 2016 ). Enamel phenotypes in M180 Tg /KO, CTRNC Tg /KO, LRAP Tg /KO, and double transgenic mice have been heterogeneous.…”

“…Enamel phenotypes in M180 Tg /KO, CTRNC Tg /KO, LRAP Tg /KO, and double transgenic mice have been heterogeneous. This is due to varying transgene dosages and suggests a cumulative effect on improving the Amelx KO enamel phenotype (Xia et al, 2016 ).…”