A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta

Abstract

Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y → H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein–protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI.

A solution NMR investigation into the impaired self-assembly properties of two murine amelogenins containing the point mutations T21→I or P41→T

“…An increased tendency to self-associate could impair amelogenesis in a number of ways. For example, premature intracellular self-association may hinder amelogenin secretion, a mechanism recently reported for the Y34→H amelogenin mutation associated with amelogenesis imperfecta [52]. Alternatively, proteolytic processing and protein-mineral interactions, both hypothesized to involve regions near the N- and C-termini of amelogenin, may be impeded in a more tightly associated complex [18,34].…”

Folding, Assembly, and Aggregation of Recombinant Murine Amelogenins with T21I and P41T Point Mutations

“…Intracellular aggregation as a result of increased self-affinity (case of T21I) or misfolding (case of P41T) could hinder amelogenin secretion. Such a mechanism was recently elucidated for the Y34H mutant in mice [Barron et al 2010]. A mutation in the trityrosyl region can also affect interactions between amelogenin and other proteins (e.g.…”

“…Two mutations in amelogenin that have been observed in humans are T21I and P40T [Lench and Winter, 1995;Collier et al, 1997;Wright, 2006], both of which cause the enamel to be hypomineralized and to have a brown discoloration. In most cases, the molecular mechanisms of defective enamel formation have still not been elucidated, although a possible molecular mechanism of the induced Y34H mutant in mice has been described recently [Barron et al, 2010].…”

Retinoic Acid Excess Impairs Amelogenesis Inducing Enamel Defects

“…Hence combinatorial deficits in enamel secretory protein expression included reductions in X-linked amelogenin ( AMELX , OMIM: 300391) (Gibson et al, 2001; Barron et al, 2010), AMBN (OMIM: 601259) (Fukumoto et al, 2004), AMTN (OMIM: 610912) (Nakayama et al, 2015), along with Odam (OMIM: 614843). These genes belong to the evolutionarily-related SCPP gene cluster, a linked group of genes also containing members regulating skeletal mineralization (Kawasaki and Weiss, 2008).…”