WDR72 models of structure and function: A stage-specific regulator of enamel mineralization

Abstract

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72−/− ameloblasts with retained proteins in the enamel matrix during maturation stage. H+/Cl− exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72−/− maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72−/− mice as compared to wildtype controls. Moreover, Wdr72−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins.

Maturation stage enamel malformations in Amtn and Klk4 null mice

“…This pattern suggests that the endocytosis mechanism, in the absence of KLK4, is still able to reabsorb much of the protein near the enamel surface, but the protein away from the surface is unable to work its way back to the cell for reabsorption. Interestingly, a pathological accumulation of enamel proteins also occurs in Wdr72 −/− mice, whose enamel shows more severe reductions in mineral density and hardness than those observed in Klk4 −/− mice [45,46]. WDR72 is an intracellular protein and defects in WDR72 contribute to the etiology of autosomal recessive amelogenesis imperfecta [47,48].…”

“…WDR72 is an intracellular protein and defects in WDR72 contribute to the etiology of autosomal recessive amelogenesis imperfecta [47,48]. Identification of proteins that co-immunoprecipitated with WDR72 suggested that WDR72 is involved in endocytosis or vesicle trafficking [45,46]. These findings, and the discovery that Klk4 is a recent (evolutionary) addition to the process of enamel maturation, suggest that endocytosis is the primary mechanism for the removal of enamel matrix proteins and that secretion of the KLK4 enzyme is a later refinement that facilitates that process by degrading extracellular proteins and thereby allowing proteins in the deeper layers to more rapidly diffuse to the enamel surface and be reabsorbed by ameloblasts.…”

Critical roles for WDR72 in calcium transport and matrix protein removal during enamel maturation

“…2012; Katsura et al. 2014). All of the mutations are located sporadically throughout the gene (exons 8, 10, 12, 15, 16, and 17) and truncate the protein (either nonsense or frameshift mutations) if translated.…”

“…2012; Katsura et al. 2014), the functions of WDR72 and its role in enamel maturation remain largely unknown. Human WDR72 encodes an intracellular protein of 1102 amino acids with no known functional domains except a β -propeller structure composed of WD40 repeat domains in its N-terminus.…”

“…Although it is suspected that WDR72 might be involved in vesicle turnover by maturation stage ameloblasts (Katsura et al. 2014), additional experimental evidence is needed before its function can be fully understood.…”