Co-Immunoprecipitation Reveals Interactions Between Amelogenin and Ameloblastin via Their Self-Assembly Domains

Abstract

Macromolecular assembly of extracellular enamel matrix proteins (EMPs) is intimately associated with the nucleation, growth, and maturation of highly organized hydroxyapatite crystals giving rise to healthy dental enamel. Although the colocalization of two of the most abundant EMPs amelogenin (Amel) and ameloblastin (Ambn) in molar enamel has been established, the evidence toward their interaction is scarce. We used co-immunoprecipitation (co-IP) to show evidence of direct molecular interactions between recombinant and native Amel and Ambn. Ambn fragments containing Y/F-x-x-Y/L/F-x-Y/F self-assembly motif were isolated from the co-IP column and characterized by mass spectroscopy. We used recombinant Ambn (rAmbn) mutants with deletion of exons 5 and 6 as well as Ambn derived synthetic peptides to demonstrate that Ambn binds to Amel via its previously identified Y/F-x-x-Y/L/F-x-Y/F self-assembly motif at the N-terminus of its exon 5 encoded region. Using an N-terminal specific anti-Ambn antibody, we showed that Ambn N-terminal fragments colocalized with Amel from secretory to maturation stages of enamel formation in a single section of developing mouse incisor, and closely followed mineral patterns in enamel rod interrod architecture. We conclude that Ambn self-assembly motif is involved in its interaction with Amel in solution and that colocalization between the two proteins persists from secretory to maturation stages of amelogenesis. Our in vitro and in situ data support the notion that Amel and Ambn may form heteromolecular assemblies that may perform important physiological roles during enamel formation.

Biomineralization of Enamel and Dentin Mediated by Matrix Proteins

“…Protein–protein interactions involving IDPs are important for biomineralization. The ability of amelogenin and ameloblastin to self-assemble and interact with each other, with cell membrane, and with the mineral is associated with their IDP character and structural flexibility (Fan et al 2009; Delak et al 2009; Bapat et al 2020). Moreover, intrinsic disorder is a unique structural feature that enables these proteins to participate in many signaling events.…”

“…Amelx null mice develop enamel with a disorganized microstructure, but the fact that enamel still forms and ribbon-like calcium phosphate crystals are still present is an indication that other enamel proteins such as enamelin and ameloblastin are intimately involved in many aspects of mineral formation, including nucleation, phase transformation, and organized growth. Our recent in vitro and in vivo studies have demonstrated that amelogenin interacts directly with enamelin and with ameloblastin (Fan et al 2009; Gallon et al 2013; Bapat et al 2020). These studies support the notion of cooperative mechanisms between enamel matrix proteins in controlling processes of crystal nucleation and growth.…”

“…( C ) Immuno-colocalization patterns (yellow) of ameloblastin (red) and amelogenin (green) within the forming enamel layer of mouse mandibular molar sections at day P5 representing transition stage. Am, ameloblasts; En, enamel; TP, Tomes’s processes (Bapat et al 2020).…”

“…3C). Using coimmunoprecipitation, we showed that ameloblastin binds to amelogenin via its previously identified, highly conserved Y/F-x-x-Y/L/F-x-Y/F self-assembly motif at the N-terminus of the region encoded by exon 5 (Wald et al 2017; Bapat et al 2020). Amelogenin and ameloblastin self-assembly motifs are therefore involved in their coassembly and can form heteromolecular assemblies in enamel extracellular space (Fig.…”

“…Amelogenin-mineral coassembly stabilizes amorphous calcium phosphate (ACP) particles (Wiedemann-Bidlack et al 2011). Nanospheres may contain enamelin and ameloblastin molecules (Fan et al 2009; Mazumder et al 2014; Bapat et al 2020). (2) Amelogenin C-terminus is cleaved by MMP-20 (Lu et al 2008).…”